There are few validated relapse prediction biomarkers for epithelial ovarian cancer (EOC). We have shown progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI) are up regulated, overexpressed survival factors in EOC. We hypothesized they would predict presence of occult EOC.
PGRN, SLPI, and the known biomarker HE4 were measured in EOC patient plasma samples, prospectively collected every 3 months from initial remission until relapse. Clinical data and CA125 results were incorporated into statistical analyses. Exploratory Kaplan-Meier estimates, dividing markers at median values, evaluated association with progression-free survival (PFS) and overall survival (OS). Area-under-the-curve (AUC) statistics were computed from receiver operating characteristic (ROC) curves to evaluate discrimination ability. A Cox proportional hazards model assessed the association between PFS, OS, and biomarkers, adjusting for clinical prognostic factors.
Samples from 23 advanced stage EOC patients were evaluated. PGRN at 3 months was the only biomarker independently associated with PFS (P<0.0001) and OS (P<0.003). When used to predict progression by 18 months, sensitivity and specificity were 93% and 100%, respectively, with AUC = 0.944. The Cox model hazard ratio for PFS, divided at 59 ng/ml by ROC analysis and adjusted for clinical factors, was 23.5 (95% CI: 2.49–220). Combinations with SLPI, HE4, and/or CA125 did not improve the model.
We report pilot data indicating a potential independent association of PGRN on EOC patient PFS and OS. A validation study will be required to confirm this finding and to inform whether PGRN warrants evaluation as a potential screening biomarker.
Progranulin (PGRN); biomarkers; progression free survival; overall survival; epithelial ovarian cancer
Recent work identified L-asparaginase (L-ASP) as a putative therapeutic target for ovarian cancer. We hypothesized L-ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L-ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion, and the assessment of sialylated proteins involved in matrix-associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell-cell and cell-matrix adhesion was observed (p<0.05 – 0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E-selectin expression was seen consistent with the unidirectional inhibitory actions observed. L-ASP concentrations were non-toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin-1, and cleavage of LC-3, indicating cell injury did occur. These data and the known mechanism of action of L-ASP on glycosylation of nascent proteins suggest that L-ASP reduction of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behavior, and causes cell injury initiating autophagy in tumor and vascular cells.
asparaginase; ovarian cancer; sialyl Lewis X; angiogenesis; autophagy
Ovarian cancer (OvCa) recurrence with development of paclitaxel resistance is an obstacle to long term survival. We demonstrated that secretory leukocyte protease inhibitor (SLPI) is a survival factor for OvCa. We hypothesize SLPI may antagonize paclitaxel injury.
Differential SLPI induction in response to paclitaxel, and response to stable forced expression of SLPI was demonstrated in A2780-1A9 cells and their paclitaxel-resistant sublines, PTX10 and PTX22 and confirmed with HEY-A8 cells. SLPI-mediated survival was reduced by the MEK inhibitor, U0126 and a humanized neutralizing monoclonal anti-SLPI antibody, CR012. OVCAR3 xenographs tested the role of CR012 in vivo.
SLPI expression was lower in A2780-1A9 OvCa cells than PTX10 and PTX22 and SLPI was induced by paclitaxel exposure. Stable SLPI expression yielded a proliferation advantage (p=0.01); expression of and response to SLPI in OVCAR3 cells was abrogated by exposure to CR012. SLPI reduced paclitaxel susceptibility of 1A9 and HEY-A8 cells (p≤0.05) and SLPI expression did not increase resistance of PTX10 and −22 cells. Both paclitaxel and SLPI overexpression induced ERK activation. Inhibition of MEK with U0126 increased paclitaxel injury and overcame SLPI-mediated cell protection. It did not reinstate PTX10 sensitivity to paclitaxel, which was associated with AKT activation. Significant inhibition of OVCAR3 xenograft growth was observed with CR012 and paclitaxel, over single agents (p≤0.001).
A two-pronged approach confirmed SLPI overcomes paclitaxel in part through activation of ERK1/2. These results credential SLPI as a molecular target for OvCa and suggest CR012 as a tool for proof of concept.
Secretory leukocyte protease inhibitor (SLPI); ovarian cancer; paclitaxel; resistance
The present study is designed to assess if exosomes released from Chronic Myelogenous Leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM-1 and VCAM-1 cell adhesion molecules and interleukin-8 expression. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE-cadherin and β-catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.
Exosomes; Chronic Myelogenous Leukemia Cells; Endothelial cells; Tumor Microenvironment
Proteomics allows characterization of protein structure and function, protein-protein interactions, and peptide modifications. It has given us insight into the perturbations of signaling pathways within tumor cells and has improved the discovery of new therapeutic targets and possible indicators of response to and duration of therapy. The discovery, verification, and validation of novel biomarkers are critical in streamlining clinical development of targeted compounds, and directing rational treatments for patients whose tumors are dependent upon select signaling pathways. Studies are now underway in many diseases to examine the immune or inflammatory proteome, vascular proteome, cancer or disease proteome, and other subsets of the specific pathology microenvironment. Successful assay verification and biological validation of such biomarkers will speed development of potential agents to targetable dominant pathways and lead to selection of individuals most likely to benefit. Reconsideration of analytical and clinical trials methods for acquisition, examination, and translation of proteomics data must occur before we march further into future of drug development.
proteomics; biomarkers; clinical trial; drug development; cancer; targeted therapy
Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI’s invasive capacity.
Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression were graded on tissue microarray and clinical samples. Statistical comparisons used unpaired T test and ANOVA, where appropriate.
SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r2=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04).
SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.
ovarian cancer; SLPI; MMP-9; metalloproteinase
To compare the recurrence-free interval (RFI), and safety profile in patients with completely resected high-risk early-stage ovarian cancer patients treated with intravenous (IV) carboplatin and paclitaxel with or without maintenance low-dose paclitaxel for 24 weeks.
Eligibility was limited to patients with Stage I-A/B (Grade 3 or clear cell), all I-C or II epithelial ovarian cancer. All patients were to receive carboplatin AUC 6 and paclitaxel 175 mg/m2 q 3 wks × 3 courses with random assignment to either observation or maintenance paclitaxel 40 mg/m2/wk × 24 wks. Recurrence required clinical or radiological evidence of new tumor.
There were 571 patients enrolled onto this study, of whom 29 were deemed ineligible due to inappropriate stage or pathology, leaving 542 patients. At least 3 cycles of treatment were administered to 524/542 (97%) of patients, and among those assigned to maintenance paclitaxel, 80% completed the regimen. The incidence of grade 2 or worse peripheral neuropathy (15.5% vs 6%), infection/fever (19.9% vs 8.7%), and dermatologic events (70.8% vs 52.1%) were higher on the maintenance regimen (p<0.001). The cumulative probability of recurring within 5 years for the maintenance paclitaxel regimen is 20% vs. 23% for surveillance (hazard ratio 0.807; 95% CI: 0.565–1.15). The probability of surviving 5 years was 85.4% and 86.2%, respectively.
Maintenance paclitaxel at 40 mg/m2/wk × 24 wks added to standard dose AUC6 and paclitaxel 175 mg/m2 × 3 doses provides no significant increase in RFI.
Background Targeting the cell-surface receptor EphA2, which is highly expressed in some solid tumors, is a novel approach for cancer therapy. We aimed to evaluate the safety profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of MEDI-547, an antibody drug conjugate composed of the cytotoxic drug auristatin (toxin) linked to a human anti-EphA2 monoclonal antibody (1C1), in patients with solid tumors relapsed/refractory to standard therapy. Methods In this phase 1, open-label study with planned dose-escalation and dose-expansion cohorts, patients received a 1-h intravenous infusion of MEDI-547 (0.08 mg/kg) every 3 weeks. Results Six patients received 0.08 mg/kg; all discontinued treatment. Dose escalation was not pursued. The study was stopped before cohort 2 enrollment due to treatment-related bleeding and coagulation events (hemorrhage-related, n = 3; epistaxis, n = 2). Therefore, lower doses were not explored and an MTD could not be selected. The most frequently reported treatment-related adverse events (AEs) were increased liver enzymes, decreased hemoglobin, decreased appetite, and epistaxis. Three patients (50%) experienced treatment-related serious AEs, including conjunctival hemorrhage, pain (led to study drug discontinuation), liver disorder, and hemorrhage. Best response included progressive disease (n = 5; 83.3%) and stable disease (n = 1; 16.7%). Minimal or no dissociation of toxin from 1C1 conjugate occurred in the blood. Serum MEDI-547 concentrations decreased rapidly, ~70% by 3 days post-dose. No accumulation of MEDI-547 was observed at 0.08 mg/kg upon administration of a second dose 3 weeks following dose 1. Conclusions The safety profile of MEDI-547 does not support further clinical investigation in patients with advanced solid tumors.
MEDI-547; EphA2; Cancer therapy; Clinical trial; Relapsed/refractory solid tumors
The power of proteomics allows unparalleled opportunity to query the molecular mechanisms of a malignant cell and the tumor microenvironment in patients with ovarian cancer and other solid tumors. This information has given us insight into the perturbations of signaling pathways within tumor cells and has aided the discovery of new drug targets for the tumor and possible prognostic indicators of outcome and disease response to therapy. Proteomics analysis of serum and ascites has also given us sources with which to discover possible early markers for the presence of new disease and for the progression of established cancer throughout the course of treatment. Unfortunately, this wealth of information has yielded little to date in changing the clinical care of these patients from a diagnostic, prognostic, or treatment perspective. The rational examination and translation of proteomics data in the context of past clinical trials and the design of future clinical trials must occur before we can march forward into the future of personalized medicine.
ovarian cancer; proteomics; clinical trials
Recent work suggested a role for NF-kB in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF-kB in ovarian cancer is unknown. We hypothesized that the NF-kB pathway is over-activated in aggressive ovarian cancers.
We assessed the levels of three NF-kB transcription factors, the activating IkB kinases and the NF-kB target MMP-9 by immunohistochemistry in ovarian cancer specimens obtained at diagnosis from a cohort of 33 patients subsequently treated with paclitaxel, cisplatin, and cyclophosphamide. Associations were made between NF-kB pathway proteins and outcome. Validation of co-expression was performed at the gene level in two independently collected cohorts of 185 and 153 ovarian cancers, respectively.
We established the presence of NF-kB proteins in newly diagnosed advanced ovarian cancers, and identified a potential association with overall survival. Transcription factors p65 and RelB were co-expressed with IKKα, one component of a key tri-molecular regulatory complex. Co-expression of the NF-kB machinery suggests activity of NF-kB signaling in these ovarian tumors. A significant association of p50 with poor overall survival was found (p=0.02). MMP9 expression showed the opposite relationship, where cases without MMP9 staining had the poorest prognosis (p=0.01), and this relationship held true at the gene expression level in an independently collected cohort of 185 ovarian cancers.
Deregulation of NF-κB activity may influence outcome in women treated with standard therapy for advanced ovarian cancer. Modification of the pathway could present an opportunity to improve outcome in the subset of women showing activity of the pathway.
NF-κB; ovarian cancer; immunohistochemistry; survival; p50; MMP9; prognosis
Carboxyamido-triazole (CAI) is a calcium influx inhibitor with anti-angiogenic and anti-invasive properties and stabilizes tumor progression in patients. We hypothesized daily oral micronized CAI with q3 week paclitaxel would be well-tolerated and active.
Twenty-nine heavily pretreated patients [median 3 (0–7)] were enrolled on five dose levels. No additive or cumulative toxicity was observed, and grade III nonhematological toxicity was rare. Neutropenia was the most common hematologic toxicity, seen in 79% of patients, with a trend towards increasing grade with higher paclitaxel doses. The recommended phase II dose defined by the maximum tolerated dose (MTD) was CAI 250 mg daily and paclitaxel 200 mg/m2 q3weeks. Pharmacokinetic analysis revealed paclitaxel increases CAI trough concentration at all dose levels by over 100% (p < 0.0001). A trend towards higher steady-state CAI trough concentrations was found in patients with a partial response (PR; p = 0.09). Six patients had confirmed PR (24%; 4–67 cycles, median 10); two patients had minor responses.
Patients and methods
Eligible patients with solid tumors received micronized CAI daily (150–250 mg PO) and paclitaxel intravenously q3weeks (175–250 mg/m2), sequentially escalating each drug. CAI preceded paclitaxel by one week to permit pharmacokinetic analysis. Patients were assessed for toxicity, pharmacokinetics and disease outcome.
The MTD of the combination of CAI and paclitaxel is 250 mg daily and 200 mg/m2 q3weeks, respectively. The combination is tolerable and has potential antitumor activity.
carboxyamidotriazole; clinical trial; ovarian cancer; paclitaxel; pharmacokinetics
The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small molecule inhibitor of IKKβ, and confirmed with RNA interference towards IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the 9-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (p=0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion. Downregulating IKKβ activity, either by a small molecule kinase inhibitor or by shRNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.
NF-κB; IKK; gene expression signature; ovarian cancer; signal transduction
To assess activity and toxicity in newly diagnosed advanced stage epithelial ovarian cancer (EOC) patients receiving dose-intense paclitaxel, cyclophosphamide, cisplatin, and filgrastim delivered with a flexible dosing schedule.
Patients with Stage III/IV EOC received cyclophosphamide 750 mg/m2, followed by 24 hr infusion of paclitaxel 250 mg/m2, and cisplatin 75 mg/m2 on day 2. Filgrastim began on day 3 at 10 μg/kg/d × 9d. Patients received six cycles of all drugs. Those with pathologic complete response or microscopic residual disease at the conclusion of six cycles of therapy received an additional cycles two to four cycles of paclitaxel with cyclophosphamide. Patients with objective response continued cyclophosphamide and paclitaxel.
62 patients were enrolled. Thirty-two of these 62 patients had stage IIIC disease, and 26 of 62 had stage IV disease. Using an intent to treat analysis, 55 (89%) experienced clinical complete remission (CCR). With a median potential follow-up of 11.4 years, the median progression free survival is 18.9 months and median survival is 5.4 years. The most serious toxicity was grade 3/4 neutropenic fever (35%). Although all participants developed peripheral neuropathy, improvement in neuropathic symptoms began with decrease or cessation of paclitaxel.
This regimen yielded a high response rate and encouraging overall survival. These data and those of the Japanese Gynecologic Oncology Group suggest that further study of dose dense or intense paclitaxel regimens in women with newly diagnosed advanced stage EOC is warranted.
ovarian neoplasms; paclitaxel; cyclophosphamide; cisplatin; antineoplastic combined chemotherapy protocols; filgrastim; drug dose-response relationship
To evaluate clinical activity and target modulation of vandetanib in women with recurrent ovarian cancer.
A phase II trial of orally administered vandetanib 300mg daily was designed to include analyses of target inhibition through paired biopsies and dynamic imaging. Core 18g needle biopsies and dynamic contrast-enhanced (DCE) MRI were obtained prior to initiation of therapy and 6wk into therapy. Biopsy samples were subjected to reverse-phase protein lysate array endpoint analysis. Cytokine concentrations were measured by ELISA in serially collected plasma samples.
Twelve patients entered the study and accrual terminated in first stage due to lack of response or disease stabilization beyond 6 months. Adverse events included rash, diarrhea, and QTc prolongation, but not hypertension. Exploratory analyses showed that EGFR phosphorylation was reduced in the 8 paired biopsy sets obtained; VEGFR2 phosphorylation was not consistently affected, nor were DCE-MRI permeability and flow parameters. Serial plasma VEGF concentrations were variable, and did not significantly change in the 11 patients assessed.
Vandetanib 300 mg daily monotherapy had no significant clinical benefit in this disease setting. Proteomic analysis of paired biopsies detected both phosphorylated-EGFR and phosphorylated-VEGFR2 in ovarian tumor tissue, but only phosphorylated-EGFR measurably inhibited by vandetanib.
ovarian cancer; vandetanib; EGFR; molecular targets; proteomics
Hypertension (HT) and hand-foot skin reactions (HFSR) may be related to the activity of bevacizumab and sorafenib. We hypothesized that these toxicities would correspond to favorable outcome in these drugs, that HT and HFSR would coincide, and that VEGFR2 genotypic variation would be related to toxicity and clinical outcomes.
Toxicities (≥ grade 2 HT or HFSR), progression-free survival (PFS), and overall survival (OS) following treatment initiation were evaluated. Toxicity incidence and VEGFR2 H472Q and V297I status were compared to clinical outcomes.
Individuals experiencing HT had longer PFS following bevacizumab therapy than those without this toxicity in trials utilizing bevacizumab in patients with prostate cancer (31.5 vs 14.9 months, n = 60, P = 0.0009), and bevacizumab and sorafenib in patients with solid tumors (11.9 vs. 3.7 months, n = 27, P = 0.052). HT was also linked to a > 5-fold OS benefit after sorafenib and bevacizumab cotherapy (5.7 versus 29.0 months, P = 0.0068). HFSR was a marker for prolonged PFS during sorafenib therapy (6.1 versus 3.7 months respectively, n = 113, P = 0.0003). HT was a risk factor for HFSR in patients treated with bevacizumab and/or sorafenib (OR(95%CI) = 3.2(1.5-6.8), P = 0.0024). Carriers of variant alleles at VEGFR2 H472Q experienced greater risk of developing HT (OR(95%CI) = 2.3(1.2 - 4.6), n = 170, P = 0.0154) and HFSR (OR(95%CI) = 2.7(1.3 - 5.6), n = 170, P = 0.0136).
This study suggests that HT and HFSR may be markers for favorable clinical outcome, HT development may be a marker for HFSR, and VEGFR2 alleles may be related to the development of toxicities during therapy with bevacizumab and/or sorafenib.
We have pioneered an in vitro pseudopod-generation model wherein suspended tumor cells are stimulated to form pseudopods into glass micropipettes in response to soluble collagen type IV (CIV). Pertussis toxin and removing intracellular calcium were found previously to be inhibitory to that process. We now extend those observations to dissect the roles of transmembrane calcium influx and circulating fatty acids on pseudopod extension. Removal of fatty acids from BSA in basal media resulted in abrogation of pseudopod formation, while reconstitution of free fatty acids restored cell pseudopod protrusion. We thus hypothesized that fatty acids may provide necessary pseudopod stimulatory signals. Addition of lysophosphatidic acid (LPA) to the fatty acid-free CIV solution or in an opposite pipette without CIV permitted approximately 50% pseudopod recovery in all pipette directions in a dose-dependent fashion. Thapsigargin (TG), an agent that releases internal calcium stores and causes opening of store-operated calcium channels, restored pseudopod protrusion up to 80% in CIV with fatty acid-free albumin. [Ca2+]i release was non-additive when cells were stimulated by TG and LPA, suggesting overlapping [Ca2+]i stores. The combination of TG and LPA in fatty acid-free albumin fully restored the pseudopod response to CIV. Addition of EGTA to chelate stimulatory media calcium blocked the pseudopod response to CIV in the presence of fatty acids. This indicates that pseudopod protrusion requires transmembrane calcium entry. Thus, extracellular lipids and calcium mobilization are required to complement CIV in pseudopod protrusion from suspended cells.
Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear.
The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines.
Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation.
This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.
The alarm anti-protease secretory leukocyte protease inhibitor (SLPI) is frequently overexpressed in ovarian cancer cells and has been proposed for inclusion in biomarker panels bits function remains unclear. We hypothesized that SLPI overexpression promotes ovarian cancer growth and survival. Low SLPI-expressing Hey-A8 ovarian cancer cells were engineered to produce functional (WT) or protease inhibitor-null (PI-) mutant SLPI; lack of PI activity was confirmed by enzymatic assay. WT/SLPI and PI- mutants stimulated significant proliferation and survival of Hey-A8 ovarian cancer cells under basal culture conditions (P≤0.02), in soft agar colony number and size (P≤0.05), and in anoikis resistance (P≤0.005). SLPI protected the ovarian cancer survival factor, progranulin (PRGN), and HEY-A8 cells from degradation and apoptosis due to neutrophil elastase. PI-/SLPI cells had greater protective activity than WT/SLPI cells. HEY-A8 murine xenografts revealed enhanced solid tumor formation, dissemination, and invasion in WT/SLPI and PI-/SLPI mutants. Increased proliferation was demonstrated by Ki-67 staining (P≤0.02). Increased secreted PRGN was seen in culture and was also observed by immunohistochemistry in the SLPI transfectant xenografts. This study describes a PI-independent function for SLPI in ovarian cancer growth and dissemination.
Sorafenib, a vascular endothelial growth factor receptor (VEGFR)-2 and RAF-kinase inhibitor, commonly causes skin toxicity. We retrospectively analyzed dermatologic toxicity in patients receiving combined anti-angiogenic therapy sorafenib and bevacizumab.
Castration-resistant prostate cancer and metastatic non-small cell lung cancer patients were accrued to phase II studies, receiving sorafenib400mg BID. A phase I study explored sorafenib 200–400mg BID with bevacizumab 5–10mg/kg every 2 weeks in patients with advanced solid tumors. Probability of development of maximum grade of dermatologic toxicity as a function of the cumulative dose of sorafenib was determined. Additional analyses compared extent of toxicity, pharmacokinetics, and patient risk factors.
Ninety-six patients were enrolled: 54 pts received sorafenib, 42 received bevacizumab/sorafenib. HFSR (hand-foot skin reaction) was observed in 50/96(52%) patients. Grade 2–3 HFSR developed in 16/54(30%) sorafenib patients and 24/42(57%) bevacizumab/sorafenib patients (p=0.012) and was associated with cumulative sorafenib exposure (p=0.0008). 24/42 phase I patients randomized to start with bevacizumab had increased risk of grade 2–3 HFSR than those starting with sorafenib (p=0.013) after adjusting for association between HFSR risk and hypertension (p=0.01), which was the only toxicity associated with HFSR. There was no association between HFSR and baseline history of neuropathy, prior taxane/platinum treatment, or systemic sorafenib levels.
Sorafenib-related HFSR is associated with increasing cumulative sorafenib dose. HFSR is increased in patients treated with bevacizumab/sorafenib combination anti-VEGF therapy, and this finding is not explained by pharmacokinetic interaction between the two agents. Our results suggest that the pathophysiology of HFSR may be related to VEGF inhibition.
sorafenib; bevacizumab; hand-foot skin reaction; rash; angiogenesis inhibition
The primary objective of this study was to evaluate the biochemical effects of gefitinib on its target signal transduction pathways in patients with relapsed epithelial ovarian cancer (EOC). The secondary aims included assessing clinical activity and toxicity, and determining the association between biochemical and clinical outcomes.
Twenty-four heavily pretreated EOC patients with good end organ function and performance status, and measurable disease were treated with gefitinib 500mg daily. Prospectively planned core tumor needle biopsies were obtained before treatment and after four weeks. Protein expression of total and phosphorylated (p) EGFR, AKT, and ERK was quantified in microdissected tumor cells using tissue lysate array proteomics.
All tumor samples had detectable levels of EGFR and pEGFR. A decrease in the quantity of both EGFR and pEGFR was seen with gefitinib therapy in over half of patients. This was not associated with clinical benefit nor were responses observed. However, trends for increased gastrointestinal and skin toxicity were seen with greater phosphorylation or quantity of EGFR, ERK, and AKT in tumor samples (p≤0.05). Gefitinib had limited clinical activity as monotherapy, in spite of documented target inhibition.
We have demonstrated gefitinib inhibits phosphorylation of EGFR in EOC tumor cells, providing proof of target in a clinical setting. Combinatorial therapy with molecular therapeutics against complementary targets may prove successful.
ovarian cancer; EGFR; gefitinib; proteomics; protein array
Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.
Staurosporine (STS) was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3E345A/D347A) within the proline-rich center of BAG3 (PXXP) and the C-terminal LEAD site (BAG3E516A/D518A). PXXP deletion mutant and BAG3E345A/D347A, or BAG3E516A/D518A respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3E516A/D518A caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.
BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.
Achieving maximal cytoreduction during surgery is a critical prognostic factor for women with advanced stage ovarian cancer. Targeting optical imaging agents directly to ovarian cancer cells by attaching them to galactosyl (galactosamine-conjugated) serum albumin, whose sugar residues bind surface lectins that are expressed in certain ovarian adenocarcinomas, may improve metastatic tumor identification and resection. Thus, we sought to demonstrate that galactosyl serum albumin-conjugated fluorophores would be a robust mechanism through which to target ovarian cancer by evaluating its tumor-targeting capability in nine human ovarian adenocarcinoma cell lines. The optical fluorophore Rhodamine Green was conjugated to galactosyl serum albumin, a non-immunogenic targeting molecule. Galactosyl serum albumin-Rhodamine Green’s ability to target nine human ovarian adenocarcinoma cell lines was evaluated by flow cytometry, fluorescence microscopy, and in vivo optical fluorescence imaging using female athymic nu/nu mice. All nine cell lines tested bound galactosyl serum albumin-Rhodamine Green more effectively than non-glycosylated controls (p<0.0001). Fluorescence microscopy demonstrated that galactosyl serum albumin-Rhodamine Green was internalized into each cell line in a galactosamine-dependent manner. In vivo optical fluorescence images of intraperitoneal tumor-bearing mice acquired three hours after intraperitoneal injection of galactosyl serum albumin-Rhodamine Green successfully differentiated between tumor and normal tissue. This technique also allowed the visualization of submillimeter-sized ovarian tumor implants. These results indicate that galactosyl serum albumin-Rhodamine Green can selectively target a variety of human ovarian adenocarcinomas for optical fluorescence imaging and thus may improve intraoperative tumor detection and resection.
Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P <0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P <0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P <0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.
Exosomes; Nanotubes; Chronic myeloid leukemia; Endothelial cells; Tyrosine kinase inhibitors