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Supplementary Figure 2 shows animal weight throughout the treatment period.

Supplementary Figure 1 shows AXL expression in a melanoma cell line panel, dose response of BGB324 and how BGB324 treatment affects invasion and migration.

Receptor tyrosine kinase AXL is found upregulated in various types of cancer, including melanoma, and correlates with an aggressive cancer phenotype, inducing cell proliferation and epithelial-to-mesenchymal transition. In addition, AXL has recently been linked to chemotherapy resistance, and inhibition of AXL is found to increase DNA damage and reduce expression of DNA repair proteins. In light of this, we aimed to investigate whether targeting AXL together with DNA damage response proteins would be therapeutically beneficial. Using melanoma cell lines, we observed that combined reduction of AXL and CHK1/CHK2 signaling decreased proliferation, deregulated cell-cycle progression, increased apoptosis, and reduced expression of DNA damage response proteins. Enhanced therapeutic effect of combined treatment, as compared with mono-treatment, was further observed in a patient-derived xenograft model and, of particular interest, when applying a three-dimensional ex vivo spheroid drug sensitivity assay on tumor cells harvested directly from 27 patients with melanoma lymph node metastases. Together, these results indicate that targeting AXL together with the DNA damage response pathway could be a promising treatment strategy in melanoma, and that further investigations in patient groups lacking treatment alternatives should be pursued.

Supplementary Figure 3 shows cell cycle profiles for Melmet 1 and WM1366 cells and YH2AX expression measured by flow cytometry for WM1366 cells. Further, the figure shows immunoblots of indicated proteins for cells treated with BGB324 and/or AZD7762 or BGB324 and/or VE-822.

Supplementary Table 1 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Supplementary Table 2 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Purpose: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM.Experimental Design: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry.Results: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, α4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products.Conclusions: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

Supplementary Tables A and B from Proteomic Analysis of Malignant Ovarian Cancer Effusions as a Tool for Biologic and Prognostic Profiling

Purpose: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics.Experimental Design: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis.Results: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P ≤ 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P ≤ 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration.Conclusions: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

The objective of this study was to analyze the expression and clinical role of mitosis regulators α-thalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC). ATRX and DAXX protein expression by immunohistochemistry was analyzed in 400 HGSC effusions. DAXX expression was additionally studied in 15 cancer cell lines, including 4 ovarian carcinoma lines, and in 81 of the 400 HGSC effusions using Western blotting. ATRX and DAXX were expressed in HGSC cells in 386/400 (96%) and 348/400 (87%) effusions, respectively. Western blotting showed DAXX expression in all 15 cell lines and in 70/81 (86%) HGSC effusions. DAXX expression by immunohistochemistry was higher in pleural compared to peritoneal effusions (p = 0.006) and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.004), and its expression was significantly associated with poor overall survival in univariate of the entire cohort (p = 0.014), as well as analysis limited to chemo-naïve effusions tapped at diagnosis (p = 0.038). The former association retained its prognostic role in Cox multivariate survival analysis (p = 0.011). ATRX expression was unrelated to clinicopathologic parameters or survival. In conclusion, DAXX is associated with disease progression and could be a prognostic marker in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Immunoregulatory protein B7-H3 is involved in the oncogenic and metastatic potential of cancer cells, as well as in drug resistance. Resistance to conventional chemotherapy is an important aspect of melanoma treatment, and a better understanding of how B7-H3 enhances drug resistance may lead to the development of more effective therapies. We investigated the in vitro and in vivo sensitivity of chemotherapeutic agents dacarbazine (DTIC) and cisplatin in sensitive and drug resistant melanoma cells with knockdown expression of B7-H3. We found that knockdown of B7-H3 increased in vitro and in vivo sensitivity of melanoma cells to the chemotherapeutic agents dacarbazine (DTIC) and cisplatin, in parallel with a decrease in p38 MAPK phosphorylation. Importantly, in B7-H3 knockdown cells we observed an increase in the expression of dual-specific MAP kinase phosphatase (MKP) DUSP10, a MKP known to dephosphorylate and inactivate p38 MAPK. DUSP10 knockdown by siRNA resulted in a reversion of the increased DTIC-sensitivity seen in B7-H3 knockdown cells. Our findings highlight the potential therapeutic benefit of combining chemotherapy with B7-H3 inhibition, and indicate that B7-H3 mediated chemoresistance in melanoma cells is driven through a mechanism involving DUSP10-mediated inactivation of p38 MAPK.

The objective of this study was to analyze the expression level and clinical role of soluble AXL (sAXL) in cancers affecting the serosal surfaces, with focus on ovarian carcinoma.sAXL protein expression by ELISA was analyzed in 572 effusion supernatants, including 424 peritoneal, 147 pleural and 1 pericardial specimens.sAXL was overexpressed in peritoneal effusions compared to pleural and pericardial specimens (p 0.001). sAXL levels were additionally significantly higher in effusions from patients with ovarian carcinoma, malignant mesothelioma and breast carcinoma compared to specimens from patients with other cancers (predominantly carcinomas of lung, gastrointestinal or uterine corpus/cervix origin) or benign reactive effusions (p 0.001). sAXL was further overexpressed in high-grade serous carcinoma (HGSC; n = 373) compared to low-grade serous carcinoma (LGSC; n = 32; p = 0.036). In HGSC, sAXL levels were significantly lower in post-chemotherapy effusions compared to primary diagnosis pre-chemotherapy specimens (p = 0.002). sAXL levels in HGSC were unrelated to chemoresponse at diagnosis, progression-free survival or overall survival. Levels were similarly unrelated to survival in LGSC and breast carcinoma.sAXL is widely expressed in malignant effusions, particularly in ovarian and breast carcinoma and in malignant mesothelioma. sAXL is overexpressed in HGSC compared to LGSC and its levels are lower following exposure to chemotherapy. However, sAXL levels are not informative of chemoresponse or survival.

Background: In precision cancer medicine, the challenge is to prioritize DNA driver events, account for resistance markers, and procure sufficient information for treatment that maintains patient safety. The MetAction project, exploring how tumor molecular vulnerabilities predict therapy response, first established the required workflow for DNA sequencing and data interpretation (2014–2015). Here, we employed it to identify molecularly matched therapy and recorded outcome in end-stage cancer (2016–2019). Material and methods: Metastatic tissue from 26 patients (16 colorectal cancer cases) was sequenced by the Oncomine assay. The study tumor boards interpreted called variants with respect to sensitivity or resistance to matched therapy and recommended single-agent or combination treatment if considered tolerable. The primary endpoint was the rate of progression-free survival 1.3-fold longer than for the most recent systemic therapy. The objective response rate and overall survival were secondary endpoints. Results: Both common and rare actionable alterations were identified. Thirteen patients were found eligible for therapy following review of tumor sensitivity and resistance variants and patient tolerability. The interventions were inhibitors of ALK/ROS1-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling for 2–3 cases each. Among 10 patients who received treatment until radiologic evaluation, 6 (46% of the eligible cases) met the primary endpoint. Four colorectal cancer patients (15% of the total study cohort) had objective response. The only serious adverse event was a transient colitis, which appeared in 1 of the 2 patients given PD-1 inhibitor with complete response. Apart from those two, overall survival was similar for patients who did and did not receive study treatment. Conclusions: The systematic MetAction approach may point forward to a refined framework for how to interpret the complexity of sensitivity versus resistance and patient safety that resides in tumor sequence data, for the possibly improved outcome of precision cancer medicine in future studies.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

Receptor tyrosine kinase AXL is found upregulated in various types of cancer, including melanoma, and correlates with an aggressive cancer phenotype, inducing cell proliferation and epithelial-to-mesenchymal transition. In addition, AXL has recently been linked to chemotherapy resistance, and inhibition of AXL is found to increase DNA damage and reduce expression of DNA repair proteins. In light of this, we aimed to investigate whether targeting AXL together with DNA damage response proteins would be therapeutically beneficial. Using melanoma cell lines, we observed that combined reduction of AXL and CHK1/CHK2 signaling decreased proliferation, deregulated cell-cycle progression, increased apoptosis, and reduced expression of DNA damage response proteins. Enhanced therapeutic effect of combined treatment, as compared with mono-treatment, was further observed in a patient-derived xenograft model and, of particular interest, when applying a three-dimensional ex vivo spheroid drug sensitivity assay on tumor cells harvested directly from 27 patients with melanoma lymph node metastases. Together, these results indicate that targeting AXL together with the DNA damage response pathway could be a promising treatment strategy in melanoma, and that further investigations in patient groups lacking treatment alternatives should be pursued.

MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent. ispartof: PIGMENT CELL & MELANOMA RESEARCH vol:33 issue:3 pages:446-457 ispartof: location:England status: published

Wee1 is a nuclear kinase regulating cell cycle progression, and has emerged as a promising therapeutic target in cancer. Expression of Wee1 has been associated with poor outcome in certain tumor types, but the prognostic impact and clinical significance in colorectal cancer is unknown. The expression of Wee1 was examined by immunohistochemistry in primary colorectal carcinomas from a prospectively collected patient cohort, and associations with clinicopathological parameters and outcome were investigated. Cell culture experiments were performed using the cell lines RKO and SW620, and the relationship with the metastasis-promoting protein S100A4 was investigated. Nuclear expression was detected in 229 of the 258 tumors analyzed (89 %). Wee1 staining was associated with low pT stage, but no other significant associations with demographic or histopathological variables were found. Moderate Wee1 staining intensity was a predictor of favorable metastasis-free and overall survival compared to strong intensity and no or weak staining. The fraction of positive cells was not a prognostic factor in the present cohort. Inhibition of Wee1 expression using siRNA or treatment with the Wee1 inhibitor MK-1775 reduced expression of the metastasis-promoting protein S100A4, but no relationship between Wee1 and S100A4 was found in the patient samples. In conclusion, Wee1 is highly expressed in primary colorectal carcinomas, but few relevant associations with clinicopathological parameters or outcome were found. The lack of clinical significance of Wee1 expression could indicate that other tumor types might be better suited for further development of Wee1 inhibitors.

Source at https://doi.org/10.1016/j.tranon.2019.04.001. Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (P ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients.

To analyze the expression and clinical role of CHK1 and CHK2 in metastatic high-grade serous carcinoma (HGSC).HGSC effusions (n = 335; 280 peritoneal, 55 pleural) were analyzed for protein expression of total CHK1 and its phosphorylated forms p-ser317 and p-ser296, as well as total CHK2 and its phosphorylated form p-thr68 using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival.Carcinoma cells stained positive, predominantly at the nuclei, in the majority of cases (range 83-100% for the five antibodies), while expression in reactive mesothelial cells and tumor-associated macrophages was more variable. Total CHK1 (p = 0.037), p-CHK1ser317 (p = 0.001), p-CHK1ser296 (p = 0.002) and p-CHK2thr68 (p 0.001) expression was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis. CHK1, p-CHK1ser296, p-CHK2thr68 and p-CHK1ser317 nuclear expression was positively related to expression of the checkpoint regulator WEE1, previously studied in this cohort (p = 0.003, p = 0.013, p = 0.001 and p = 0.01, respectively). Higher total CHK1 (p = 0.007), p-CHK1ser317 (p = 0.004), CHK2 (p = 0.01) and p-CHK2thr68 (p = 0.048) expression was significantly related to shorter overall survival in univariate analysis, and CHK1ser317 was an independent prognostic marker in multivariate analysis (p = 0.025). Higher p-CHK1ser317 (p = 0.03) and CHK2 (p = 0.034) expression was additionally associated with poor progression-free survival.CHK1 and CHK2 and their activated forms are frequently expressed in HGSC effusions, with higher expression following exposure to chemotherapy, and their expression is related to survival.

The aim of the present study was to investigate the expression and clinical role of the aurora A and aurora B kinases in primary and metastatic serous ovarian carcinoma. AURKA and AURKB messenger RNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction. Aurora A and aurora B protein expression by immunohistochemistry was additionally analyzed in 147 tumors. Messenger RNA and protein expression at different anatomical sites were studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. AURKA and AURKB messenger RNA and their protein product were demonstrated in all primary carcinomas, solid metastases, and effusions. The expression of AURKA messenger RNA and aurora A protein was higher in effusions compared with solid specimens (P = .003 and P = .006, respectively). AURKB messenger RNA expression was higher in primary carcinomas, and solid metastases obtained prechemotherapy compared with postchemotherapy (P.001 and P = .012, respectively), with no such difference in effusions (P.05). Low aurora B protein expression was associated with primary chemotherapy resistance (P = .006) and poor treatment response (P = .013) in prechemotherapy effusions. No significant association was found between messenger RNA levels or protein expression and progression-free or overall survival. The present study documents for the first time frequent aurora A and aurora B expression in metastatic ovarian carcinoma, suggesting a role in cancer progression, with higher aurora A expression in effusions compared with primary carcinomas and solid metastases. Low AURKB messenger RNA expression in prechemotherapy effusions might be predictive of intrinsic chemotherapy resistance.

Cytoreductive surgery and intraperitoneal (i.p.) chemotherapy constitute a curative treatment option in mucinous peritoneal surface malignancies of intestinal origin, but treatment outcome is highly variable and the search for novel therapies is warranted. Immunotoxins are attractive candidates for targeted therapy in the peritoneal cavity because of direct cytotoxicity, distinct mechanisms of action and tumor cell selectivity. The MOC31PE immunotoxin targets the tumor-associated adhesion protein EpCAM (Epithelial Cell Adhesion Molecule), and has been administered safely in early clinical trials. In our work, the efficacy of i.p. administration of MOC31PE alone and together with mitomycin C (MMC) was investigated in unique animal models of human mucinous peritoneal surface malignancies. In initial model validation experiments, clear differences in efficacy were demonstrated between MMC and oxaliplatin, favoring MMC in five investigated tumor models. Subsequently, MOC31PE and MMC were given as single i.p. injections alone and in combination. In the PMCA-2 model, moderate growth inhibition was obtained with both drugs, while the combination resulted in at least additive effects; whereas the PMP-2 model was highly sensitive to both drugs separately and in combination and intermediate sensitivity was found for the PMCA-3 model. Furthermore, results from ex vivo experiments on freshly obtained mucinous tumor tissue from animals and patients suggested that classic mechanisms of immunotoxin activity were involved, i.e., inhibition of protein synthesis and induction of apoptosis. The present results suggest that adding MOC31PE to MMC-based i.p. chemotherapy should be further explored for EpCAM-expressing peritoneal surface malignancies, and a phase I trial is in preparation.

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

To investigate the expression of MGST1 in primary tumors, solid metastases and metastatic effusions in advanced-stage serous ovarian carcinoma (OC) and analyze the association with clinicopathologic parameters, including chemotherapy resistance and survival.MGST1 mRNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, 52 solid metastases) from 144 patients using real-time quantitative PCR (qRT-PCR). Forty-two of the 88 effusions were additionally analyzed for MGST1 protein expression by Western blotting.mRNA expression of MGST1 was higher in primary carcinomas and solid metastases compared to effusions (p=0.008 and p=0.012, respectively). In patient-matched samples, mRNA expression of MGST1 was higher in solid metastases compared to effusions (p=0.023), and a trend for higher MGST1 levels in solid metastases compared to primary tumors was observed (p=0.06). Biopsies from primary carcinomas obtained from patients with200 ml ascites at diagnosis had higher mRNA expression of MGST1 compared to samples from patients with200 ml ascites (p=0.037). MGST1 mRNA expression was not associated with age, histological grade, tumor stage, residual disease volume, response to chemotherapy, chemotherapy resistance or survival. Western blot analysis of patient-matched effusions showed high concordance between MGST1 protein and mRNA levels measured by qRT-PCR (p0.001).The present study documents frequent MGST1 mRNA and protein expression in OC. The data suggest increased activity of oxidative response pathways, reflected by higher mRNA expression, in solid OC tumors compared to metastatic effusions. Additionally, a tumor microenvironment consisting of ascites may induce antioxidant activity.

The objective of this study was to analyze the expression and clinical role of the high mobility group AT hook (HMGA) protein in advanced-stage serous ovarian carcinoma. HMGA2 protein expression was investigated in 199 effusions and in 50 patient-matched primary tumors and solid metastases using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. HMGA2 was expressed in tumor cells in 94.5 %, 96 %, and 90 % of specimens, respectively. There was no difference in HMGA2 expression between patient-matched samples from different anatomic sites (p > 0.3). HMGA2 expression in chemo-naive samples was significantly higher in older patients (p = 0.006, p = 0.01, and p = 0.005 for effusions, primary tumors, and solid metastases, respectively). No association was found with residual disease volume. Furthermore, HMGA2 expression was not associated with FIGO stage (p > 0.2), except in chemo-naive effusions (n = 106, p = 0.016). There was no difference in HMGA2 expression between chemo-naive samples and samples obtained post-chemotherapy in effusions (p = 0.2) or primary tumors (p = 0.1). However, solid metastases obtained after chemotherapy exposure had higher HMGA2 expression compared with chemo-naive samples (p = 0.032). HMGA2 expression was unrelated to chemotherapy response or survival. However, it was directly related to protein expression of the previously studied cancer stem cell marker Nestin (p = 0.01) and the gap junction protein claudin-7 (p = 0.02) and inversely related to the mRNA level of the E-cadherin repressor SIP1 (p = 0.02). This study provides evidence that HMGA2 is universally expressed in advanced-stage ovarian serous carcinoma irrespective of anatomic site, suggesting that HMGA2 may have a clinical role as therapeutic target.

We aimed to identify primary platinum resistance in epithelial ovarian cancer (OC) patients with FIGO stage III–IV disease by an in vitro drug-response assay and to correlate the findings with clinical response. We considered whether neoadjuvant chemotherapy or anatomic sample site and tumor heterogeneity would influence the results. We combined the ATP-based tumor-chemosensitivity and the extreme drug resistance assays for testing of 85 biopsies from 58 patients. Tumors were classified as sensitive or resistant by a resistance index (RI). We did separate analyses of primary tumors and metastases and compared chemo-naive samples with samples obtained after neoadjuvant chemotherapy. Results were analyzed for association with clinical platinum resistance, progression-free survival (PFS), and overall survival (OS). RI ≤ 250 predicted primary platinum resistance, without misclassification of sensitive patients. The test sensitivity for primary tumors was 15/15, specificity 3/10, negative predictive value 3/3, and positive predictive value 15/22. Patients with in vitro platinum-resistant samples had shorter PFS compared with patients with sensitive samples (3.4 vs. 10.0 months, p = 0.02). Comparing patient-matched primary and metastatic samples, there was about 1/3 mismatch in resistance. RI for platinum was lower in primary tumors exposed to neoadjuvant chemotherapy than in chemo-naive tumors (p

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Florenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c-FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c-FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c-FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal-to-noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c-FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c-FLIP by Abcam and the IntraStain kit by Dako performed best. c-FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c-FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c-FLIP levels and expression of the previously studied apoptosis marker cleaved caspase-3 (P = 0.029). Conclusions: An assay for measuring c-FLIP in cytology specimens is presented. c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.

The objective of this study was to analyze the clinical role of nestin, a stem cell marker, and class III β-tubulin in advanced-stage serous ovarian carcinoma. Nestin and class III β-tubulin protein expression were investigated in 217 effusions using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters including chemotherapy response and survival. Class III β-tubulin and nestin were expressed in tumor cells in 98.6% and 95.6% of specimens, respectively. Staining extent was comparable in prechemotherapy and postchemotherapy effusions. No association was found with patient age, histologic grade, International Federation of Gynecology and Obstetrics stage, primary surgery versus secondary debulking, or residual disease volume. High class III β-tubulin expression in prechemotherapy effusions was significantly associated with primary chemoresistance (progression-free survival

The insulin like growth factor (IGF) signaling pathway has been shown to contribute to melanoma progression, but little is known about the role of the IGF binding protein 3 (IGFBP-3) in melanoma biology. The aim of the present study was to characterize expression, function and regulation of IGFBP-3 in malignant melanomas and study its potential as a biomarker. The expression of IGFBP-3 varied between different human melanoma cell lines and reintroduction of the protein in non-expressing cells led to induction of apoptosis. Interestingly, in cell lines expressing endogenous IGFBP-3, siRNA silencing of the protein led to a cell line-dependent decrease in proliferation, but had no effect on apoptosis and invasion. Examination of patient material showed that IGFBP-3 is unexpressed in benign nevi while a slight increase in protein expression was seen in primary and metastatic melanoma. However, expression of the protein was low and no correlation was found with circulating levels of IGFBP-3 in serum, suggesting that IGFBP-3 has limited potential as a predictive marker in malignant melanoma. We showed that promoter methylation of IGFBP-3 occurred in both melanoma cell lines and patient material, implicating epigenetic silencing as a regulation mechanism. Furthermore, expression of the protein was shown to be regulated by the PI3-kinase/AKT and MAPK/ERK1/2 pathways. In summary, our findings suggest that IGFBP-3 can exert dual functional effects influencing both apoptosis and proliferation. Development of resistance to the antiproliferative effects of IGFBP-3 may be an important step in progression of malignant melanomas.

Objective: Seprase, dipeptidyl peptidase IV (DPPIV) and urokinase-type plasminogen activator (uPA) play a crucial role in the degradation of the extracellular matrix and in the progression of various human tumors. However, their pathophysiologic significance in esophageal carcinoma has not yet been fully elucidated. Methods: The expression of seprase, DPPIV and uPA in esophageal dysplasia, squamous cell carcinoma (SCC) and normal epithelium was examined by immunohistochemistry. Results: Seprase, DPPIV and uPA immunoreactivity was found in dysplastic and cancer cells as well as in stromal cells adjacent to dysplasia and cancer sites, but not in normal epithelium. We found a significant association between uPA expression and sex, tumor size and histological classification in carcinomas. High expression of DPPIV in cancer cells correlated with longer survival of the patients. No significant associations between seprase and clinicopathological features either in dysplasia or in carcinomas were found. Finally, we demonstrated higher levels of seprase, DPPIV and uPA in SCC cell lines than in normal esophageal epithelial cell lines. Conclusions: Our results showed that seprase, DPPIV and uPA are expressed in both premalignant and malignant forms of SCC, but are lacking in normal esophageal epithelia, suggesting that they are involved in SCC neoplastic progression.

Summary The objective of this study was to analyze the clinical role of 9 microRNAs (miRs) previously found to be overexpressed in ovarian carcinoma effusions compared with primary ovarian carcinomas. High-grade serous carcinoma effusions (n=148) were analyzed for expression of miR-29a, miR-31, miR-99b, miR-182, miR-210, miR-221, miR-222, miR-224, and miR-342 using quantitative polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters and survival. miR-29a and miR-31 levels were further assessed for association with protein expression of their targets Stathmin and DNA methyltransferase-3A (DNMT3A) by immunohistochemistry and Western blotting, respectively. miRNA levels were unrelated to clinicopathological parameters. However, higher miR-29a levels were significantly related to longer overall survival in univariate ( P =.007) and Cox multivariate survival analysis ( P =.045). miR-29a levels were inversely related to those of its target DNMT3A ( P =.048), and higher DNMT3A expression was significantly related to poor overall survival in univariate ( P =.03) and Cox multivariate ( P =.016) survival analysis. In contrast, miR-31 levels were directly related to cytoplasmic phospho-Stathmin expression ( P =.029) and unrelated to Stathmin and nuclear phospho-Stathmin, and both Stathmin and phospho-Stathmin expressions were unrelated to survival. miR-29a and its target DNMT3A are novel candidate biomarkers of longer and shorter survival, respectively, in metastatic high-grade serous carcinoma.

Inhibitor-of-apoptosis proteins (IAPs) mediate cancer cell survival and chemoresistance. We analyzed the expressions of X-linked IAP (XIAP), survivin, and livin in malignant mesothelioma. Ten effusions were analyzed for XIAP, survivin, and livin expression using immunoblotting. Based on the immunoblotting results, 112 mesotheliomas from 94 patients (pleural, n = 77; peritoneal, n = 35; solid, n = 68; effusions, n = 44) were immunostained for XIAP and survivin expression. Results were analyzed for associations with anatomic site (pleura versus peritoneum), specimen type (solid versus effusion), proliferation (Ki-67 score), and survival. Immunoblotting showed expression of XIAP in 9 of 10 effusions and that of survivin in 4 of 10 effusions, but no expression of livin. Immunohistochemistry showed cytoplasmic XIAP expression in 71 of the 112 (63%) tumors. XIAP expression was significantly higher in peritoneal mesotheliomas than in pleural mesotheliomas (P = .001) and in effusions than in solid lesions (P = .017). Cytoplasmic survivin was found in 75 of the 112 (67%) tumors and showed no site-related difference. Nuclear survivin was expressed in 37 of the 112 (33%) tumors, with a trend for positive association with the Ki-67 score (P = .051). Nuclear survivin (P = .003) and Ki-67 (P = .013) were downregulated in effusions as compared with solid tumors. Higher XIAP expression and Ki-67 score were associated with a trend for poor overall survival (P = .064 for both) in the univariate analysis. XIAP and survivin, but not livin, are frequently expressed in malignant mesotheliomas. Nuclear survivin expression is reduced in effusions as compared with solid lesions concomitantly with reduced proliferation. XIAP is upregulated in mesothelioma effusions and peritoneal mesotheliomas, suggesting a prosurvival role in malignant mesothelioma cells, particularly at these anatomic sites.

Background Malignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets. Methods In the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models. Results Our in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts. Conclusions These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1474-8) contains supplementary material, which is available to authorized users.

We evaluated 123 formalin-fixed, paraffin-embedded samples, including neuroendocrine tumors, adult brain, mesonephric tissues, and from various other sites. A pre-B lymphoma cell line, Daudi, and a small cell carcinoma cell line, NCL-H128, were evaluated by Western blot. All tissues were immunostained by mouse monoclonal anti-Pax-5 antibody by using standard, synthetic polymer-based detection methods. Our study describes for the first time distribution of Pax-5 in adult brain tissue, including periaqueductal gray matter of the midbrain, area postrema of the medulla oblongata, and occasional cells of the spinal trigeminal nucleus (caudal nucleus). We confirm that Pax-5 is expressed regularly in poorly differentiated neuroendocrine tumors but never in well-differentiated classic carcinoid tumors. Pax-5 expression also was found readily in benign and malignant mesonephric tissues and focally in mullerian duct-derived tissues and tumors. Expression of Pax-5 in the Daudi and NCL-H128 cell lines was confirmed by Western blot. Together, these results are important for correct interpretation of results in immunophenotyping of undifferentiated tumors, for diagnosis of mesonephric carcinoma, and, potentially, for correct classification of neuroendocrine tumors in small biopsy samples.

The aim of the present study was to evaluate HLA-G expression in breast carcinoma and malignant mesothelioma (MM). Malignant breast carcinoma effusions (46) and corresponding solid tumors (39) and 104 MM (26 effusions, 78 solid tumors) were analyzed using immunohistochemistry (IHC). HLA-G protein and mRNA expression were further studied using immunoblotting (IB) and RT-PCR. HLA-ABC expression was analyzed using flow cytometry (FCM). IHC showed predominantly focal HLA-G expression in 12 of 46 (26%) breast carcinoma effusions and 16 of 39 (41%) solid lesions. In MM, 20 of 78 (26%) solid lesions and 14 of 26 (54%) effusions were focally HLA-G positive. Expression in MM was higher in effusions (p=0.008). IB showed more frequent HLA-G expression in MM compared with breast carcinoma effusions, while RT-PCR showed HLA-G mRNA expression in both tumors. FCM showed conserved HLA-ABC expression in 15 of 15 effusions. Breast cancer patients with HLA-G-positive tumor cells had shorter disease-free survival (mean 37 vs 85, median 25 vs 31 months), though not significantly (p=0.14). In conclusion, HLA-G is focally expressed in MM and breast carcinoma, while HLA-ABC expression is conserved. However, the up-regulated expression of HLA-G in MM effusions and its possible association with shorter disease-free survival in advanced stage of breast carcinoma suggest a possible role in immune response evasion in some tumors.

To analyze the expression of the AP-2gamma transcription factor in ovarian borderline tumors, early-stage ovarian carcinoma and advanced-stage ovarian carcinoma, and to evaluate its prognostic role in advanced-stage tumors.Sections from 14 normal ovaries, 75 borderline tumors, 22 FIGO stage I invasive ovarian carcinomas, and 306 advanced-stage (FIGO stages II-IV) ovarian carcinomas (42 primary tumors, 62 solid metastases, 202 effusions) were evaluated for expression of the transcription factor AP-2gamma using immunohistochemistry. Sixty-three effusions and two cell lines (SKOV-3 and OVCAR-3) were additionally studied using immunoblotting. The prognostic role of AP-2gamma in advanced-stage carcinomas was analyzed.AP-2gamma was detected in the nucleus of tumor cells in 28/75 (37%) borderline tumors, 13/22 (59%) FIGO stage I carcinomas, and 255/306 (83%) advanced-stage carcinomas (P0.001, Chi-square test). Benign ovaries were uniformly negative. Expression was largely limited to carcinoma cells in effusions. Solid lesions and effusions from advanced-stage carcinomas showed comparable expression. Immunoblotting showed AP-2gamma expression in 59/61 effusions and both cell lines. AP-2gamma expression did not correlate with survival.AP-2gamma expression is upregulated in advanced-stage ovarian carcinoma compared to early-stage carcinomas, borderline tumors, and the ovarian surface epithelium, and AP-2gamma is specifically localized to cancer cells in effusions, suggesting a role in tumor progression. The lack of predictive value for this transcription factor in advanced-stage disease may be related to its frequent expression.

Purpose: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics. Experimental Design: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis. Results: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P ≤ 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P ≤ 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration. Conclusions: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

Our purpose was to analyze, by immunohistochemistry, the expression of activated serine-threonine protein kinase B (p-Akt) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in benign nevi and primary and metastatic melanomas and to correlate the expression level with clinical variables. We observed cytoplasmic and/or nuclear expression of p-Akt in 22 (54%) of 41 benign nevi, 112 (71.3%) of 157 primary tumors, and 50 (71%) of 70 metastases. Cytoplasmic PTEN staining was observed in 0 (0%), 152 (87.7%), and 64 (90%) of 41 nevi, 162 primary tumors, and 71 metastases, respectively. A significant positive correlation was seen between PTEN and p-Akt cytoplasmic expression (P < .001) in primary melanomas. Cytoplasmic p-Akt expression showed a positive association with cyclin A in superficial spreading (P = .038) but not in nodular (P = .22) melanomas. Cytoplasmic p-Akt and PTEN expression did not have an impact on disease-free and overall survival, but complete lack of nuclear p-Akt expression was a predictor of shorter disease-free survival (P = .025) for patients with superficial spreading melanoma. The coordinated balance between cell division, growth, and programmed cell death (apoptosis) maintains cellular homeostasis, and disturbance of this balance might result in cancer development. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to have an important role in the regulation of the aforementioned processes, and dysregulation of this pathway is a common feature of many cancers, including melanoma (reviewed by Vivanco and Sawyers 1 ).

The molecular biology underlying the development of highly malignant peripheral nerve sheath tumors (MPNSTs) remains mostly unknown. In the present study, the expression pattern of 10 selected cell cycle components is investigated in a series of 15 MPNSTs from patients with (n = 9) or without (n = 5) neurofibromatosis type 1 (NF1). Thirteen tumors did not express the cyclin-dependent kinase inhibitor, p16(INK4A), an observation that was related to homozygote gene deletions in three tumors, heterozygote deletions in five, and gross gene rearrangements in five. The absence of protein expression in the tumors with one seemingly intact allele was not caused by promoter hypermethylation of p16(INK4A) or p14(ARF). All tumor samples expressed normal sized RB1, cyclin D3, CDK2, CDK4, p21(CIP1), and p27(KlP1) proteins, and only a single tumor showed an aberrant protein band for one of these proteins, p21(CIP1). Cyclin D1 was absent in four tumors; all except one tumor showed expression of TP53 protein, and three of nine MPNSTs had expression of normal-sized MDM2. In conclusion, this study shows that the vast majority of MPNSTs had gross rearrangements of the p16(INK4A) gene, explaining the absence of the encoded protein in the same tumors. The level of expression was equally distributed between the familial (NF1) and sporadic cases, although it should be noted that the 2 cases with p16(INK4A) expression were sporadic. The data imply that the complete absence of p16(INK4A) is sufficient for activation of the cell cycle in most MPNSTs; thus, it is not necessary for tumor proliferation to further stimulate the cycle through alteration of other central components.

Ets-1 transcription factor has been associated with tumor progression in various carcinomas, but its expression in malignant melanoma was only recently described. The study was conducted in two steps: exploratory and confirmatory. In the first step, we studied 69 primary melanomas, 28 metastatic melanomas, 10 usual intradermal nevi and 13 various melanocytic skin lesions. In the second step, an additional group of 98 patients with follow-up of up to 200 months was also evaluated. Immunohistochemical analysis of formalin-fixed/paraffin-embedded tissues was performed using 1G11 antibody and polymer conjugate for visualization. While Ets-1 was variably expressed in 83% primary melanomas in exploratory and 69% in the confirmatory group, the expression of Ets-1 was also found in normal benign melanocytes and all nevi. Analysis of the exploratory group revealed lower expression of Ets-1 in primary melanomas than in common nevi (P=0.048, Mann-Whitney U-test) and metastatic melanomas expressed significantly less Ets-1 than primary melanomas (P=0.015, Mann-Whitney U-test). There was a negative correlation between Ets-1 expression and the largest dimension of the primary tumors (r=0.23, P=0.034, Spearman's correlation rank test), but no correlation with the depth of tumor invasion (Breslow thickness) or the presence of ulceration was found. Analyses of the confirmatory group revealed no association between Ets-1 expression with disease-specific survival or time to treatment failure. However, a statistical trend was found for worse outcome for those primary melanomas that had strong expression (H-score >100) of Ets-1 (P=0.054). Ets-1 is expressed in benign melanocytes probably due to their neural crest origin. We conclude that Ets-1 expression cannot be used to differentiate between benign and malignant melanocytic lesions and it has no definite association with clinical outcome. At the same time, its role in tumor progression in some cases of malignant melanoma cannot be entirely excluded.

The aim of the present study was to analyze the expression of S100A4 and E-cadherin in a panel of primary and metastatic malignant melanoma, and to correlate the expression level to clinicopathological parameters. The expression of S100A4 was examined by immunohistochemistry in 99 superficial spreading and 60 nodular primary melanomas, while the expression of E-cadherin was analyzed in 92 superficial spreading and 52 nodular lesions from the same panel. The expression levels of S100A4 and E-cadherin in the biopsies were inversely correlated, with S100A4 being expressed at the highest frequency in the nodular and E-cadherin in the superficial spreading lesions, respectively. When analyzing the melanoma subgroups separately, it was revealed that expression of S100A4 had a more significant impact on patient outcome in early superficial spreading melanomas than in the nodular subtype, while E-cadherin expression did not predict patient outcome in any of the subgroups. When examining all the patients, both markers give clinical information as predictors for disease-free survival, but when combining the expression of the two markers, a stronger significant correlation between high E-cadherin expressing/S100A4 negative biopsies and increased disease-free survival (P=0.002) was revealed, demonstrating the importance of examining the expression of more than one factor involved in the metastatic cascade when predicting patient outcome. We have also evaluated the relationship between the expression of these two antigens and cell cycle and signal transduction factors.

In the present study the deacetylase inhibitor trichostatin A (TSA) was used to elucidate the effect of protein acetylation on cell cycle progression and survival in seven human malignant melanoma cell lines. It was shown that TSA treatment led to a transient G(2)/M phase delay and accumulation of unphosphorylated retinoblastoma protein (pRB) in all cases. TSA significantly induced protein expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in a dose-dependent manner in all cell lines including those not expressing p21(WAF1/CIP1) constitutively, whereas the levels of both wild-type and mutated p53 protein were reduced. The effect on p53 was not a direct result of inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) activation by TSA, as treatment of the cells with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEK1) inhibitor PD98059 did not result in decreased p53 protein level. Furthermore, TSA treatment led to reduction in cyclin D1 whereas cyclin D3 accumulated, the latter due to increased protein stability. Similarly, cyclin A protein was reduced whereas cyclin E level was elevated. The effect on p27(Kip1), CDK4 and CDK2 was only marginal. In all the examined cell lines, TSA treatment resulted in a profound induction of apoptosis and cleavage of poly-(ADP-ribose)-polymerase (PARP) indicative of caspase activity. Similarly, TSA-mediated apoptosis was reversed by the caspase-inhibitor z-vad-fmk. Altogether, these results suggest that p21(WAF1/CIP1) in melanomas is silenced by deacetylation, and furthermore that inhibition of deacetylation may have potential in anticancer therapy of melanoma patients.

BACKGROUND The granulin-epithelin precursor (GEP) was preferentially expressed in invasive ovarian tumor epithelium specimens compared with specimens of borderline ovarian tumors. The objective of the current study was to evaluate the anatomic site-related and cellular expression of GEP and its association with clinicopathologic parameters and survival in patients with advanced-stage ovarian carcinoma. METHODS Effusions (n = 190), corresponding primary tumor specimens (n = 64), and specimens of metastatic lesions (n = 125) were analyzed using immunohistochemistry with a specific polyclonal antipeptide antibody. In addition, 36 effusions were analyzed using immunoblotting. RESULTS GEP was detected in tumor cells in 171 of 190 (90%) effusions and demonstrated both focal membrane and cytoplasmic localization. Mesothelial cells were often GEP positive (81%). GEP was found in carcinoma cells in 180 of 189 (95%) tumor biopsy specimens, with stromal and endothelial cell expression in 93 of 180 (52%) and 124 of 185 (67%) specimens, respectively. Lower GEP expression in stromal cells was observed in metastases sampled during or after chemotherapy (P = 0.034). The presence of GEP-positive stromal cells in untreated primary tumor specimens correlated with worse overall survival (P = 0.014). Significantly more frequent GEP expression was observed in tumor cells of both primary (P = 0.002) and metastatic (P < 0.001) tissue specimens compared with malignant effusions. CONCLUSIONS GEP expression was observed in primary and metastatic epithelial ovarian carcinoma specimens, with down-regulated expression in tumor cells of malignant effusions. The poor outcome associated with stromal GEP expression suggests a prognostic role for this growth factor in ovarian carcinoma. Cancer 2004. Published 2004 American Cancer Society.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.