Higher Levels In Tumor Tissues Research Articles

Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer.

Aberrant protein post-translational modification is a hallmark of malignant tumors. Lysine succinylation (Ksucc) plays a vital role in cell energy metabolism in various cancers. However, whether succinylation can be catalyzed by acetyltransferase p300 remains unclear. In this study, we unveiled that p300 is a "writer" for succinylation, and p300-mediated Ksucc promotes cell glycometabolism in lung adenocarcinoma (LUAD). Specifically, our succinylome data revealed that EP300 deficiency leads to the systemic reduction of Ksucc, and 79.55% of the p300-succinylated proteins were found in the cytoplasm, which were primarily enriched in the carbohydrate metabolism process. Interestingly, deleting EP300 led to a notable decrease in Ksucc levels on several glycolytic enzymes, especially Phosphoglycerate Kinase 1 (PGK1). Mutation of the succinylated site of PGK1 notably hindered cell glycolysis and lactic acid excretion. Metabolomics in vivo indicated that p300-caused metabolic reprogramming was mainly attributed to the altered carbohydrate metabolism. In addition, 89.35% of LUAD patients exhibited cytoplasmic localization of p300, with higher levels in tumor tissues than adjacent normal tissues. High levels of p300 correlated with advanced tumor stages and poor prognosis of LUAD patients. Briefly, we disclose the activity of p300 to catalyze succinylation, which contributes to cell glucose metabolic reprogramming and malignant progression of lung cancer.

Systematic characterization of m6A proteomics across 12 cancer types: a multi-omics integration study.

The modification patterns of N6-methyladenosine (m6A) regulators and interacting genes are deeply involved in tumors. However, the effect of m6A modification patterns on human proteomics remains largely unknown. We evaluated the molecular characteristics and clinical relevance of m6A modification proteomics patterns among 1013 pan-cancer samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). More than half of the m6A proteins were expressed at higher levels in tumor tissues and presented oncogenic characteristics. Furthermore, we performed multi-omics analyses integrating with transcriptomics data of m6A regulators and interactive coding and non-coding RNAs and developed a m6A multi-omics signature to identify potential m6A modification target proteins across global proteomics. It was significantly associated with overall survival in nine cancer types, tumor mutation burden (P = 0.01), and immune checkpoints including PD-L1 (P = 4.9 × 10-8) and PD-1 (P < 0.01). We identified 51 novel proteins associated with the multi-omics signature (PFDR < 0.05). These proteins were functional through pathway enrichment analyses. The protein with the highest hit frequency was CHORDC1, which was significantly up-regulated in tumor tissues in nine cancer types. Its higher abundance was significantly associated with a poorer prognosis in seven cancer types. The identified m6A target proteins might provide infomation for the study of molecular mechanism of cancer.

SR-BI expression regulates the gastric cancer tumor immune microenvironment and is associated with poor prognosis

<b>Aim:</b> Scavenger receptor class B, type I (SR-BI) is an integral plasma membrane protein that has been reported to be overexpressed in various malignancies, such as renal cancer, breast cancer, and prostate cancer, and is an independent prognostic factor. However, the clinical value and expression of SR-BI in GC are unknown. Our research aimed to explore the role of SR-BI in combination with immune markers as a diagnostic and prognostic marker for gastric cancer (GC). <b>Methods:</b> GC tissues, paracancerous tissues, and clinicopathological data of 149 patients were collected. The expression level of SR-BI, Tumor-infiltrating lymphocytes (TILs), and PD-L1 were evaluated by immunohistochemistry (IHC). The associations of the SR-BI staining intensity with clinicopathological features and immune markers were determined by the chi-square test. Univariate and multivariate COX regression analyses were used to evaluate independent prognostic factors. Kaplan–Meier analyses were performed to plot the survival curve. <b>Results:</b> Our results indicated that SR-BI was expressed at higher levels in tumor tissues than in adjacent paracancerous tissues (<i>p</i> < 0.001), and patients with high levels of SR-BI expression had a worse prognosis. Univariate and multivariate analyses revealed that high SR-BI expression was an independent factor for poor prognosis. The chi-square test determined that the expression of SR-BI was negatively correlated with CD4+ T cells and CD8+ T cells (CD4+ T cells, <i>p</i> = 0.013; CD8+ T cells, <i>p</i> = 0.021), and positively correlated with PD-L1 (<i>p</i> = 0.022). Finally, survival analysis revealed that CD4+ T cells were associated with the prognosis of GC patients (<i>p</i> = 0.019), and the combined survival analysis of SR-BI and CD4+ T cells was also statistically significant (<i>p</i> = 0.030). <b>Conclusion:</b> SR-BI is highly expressed in GC tissue and associated with poor prognosis. Moreover, SR-BI can also regulate the GC tumor immune microenvironment.

MLN2238 exerts its anti-tumor effects via regulating ROS/JNK/mitochondrial signaling pathways in intrahepatic cholangiocarcinoma.

Background: Intrahepatic Cholangiocarcinoma (iCCA) is a highly malignant tumor with limited treatment options that contributes largely to cancer-related deaths worldwide. Compared with traditional transcriptomic analysis, single-cell RNA sequencing (scRNA-seq) is emerging as a more advanced and popular tool for the in-depth exploration of cellular diversity and molecular complexity. As a next-generation proteasome inhibitor, MLN2238 presents better pharmacodynamics, pharmacokinetics, and therapeutic responses in various cancers. However, its effects and mechanisms of action in iCCA remain unknown. Methods: iCCA tumor heterogeneity was determined based on 4,239 qualified scRNA-seq data from 10 iCCA samples. The potential biological roles of proteasome-related genes in iCCA were investigated using a pseudo-trajectory reconstruction. The effect of MLN2238 on iCCA cell proliferation was estimated using the CCK-8, EdU, and clone formation assays. Flow cytometry was used to examine the effect of added MLN2238 on cell cycle and apoptosis levels. Autophagic flux was detected using AdPlus-mCherry-GFP-LC3B cells. ROS levels and mitochondrial membrane potential were determined using DCFH-DA probing and JC-1 staining. JNK activation and mitochondrial apoptosis were observed using western blotting and immunofluorescence microscopy, respectively. Finally, we used a tumor-bearing mouse model to validate its efficacy in vivo for iCCA treatment. Results: Proteasome-related genes were dysregulated in iCCA progression and expressed at higher levels in tumor tissues. MLN2238 suppressed cell proliferation, blocked the cell cycle in the G2/M phase, promoted apoptosis, and induced cytoprotective autophagy in iCCA cells. Furthermore, MLN2238 increased ROS levels and activated the JNK signaling pathway. Inhibition of ROS and JNK activation by NAC and SP600125 significantly reversed MLN2238-induced apoptosis. MLN2238 also suppressed the growth of iCCA tumors in vivo. Conclusion: Proteasome-related genes play pivotal roles in iCCA development. MLN2238, as a proteasome inhibitor, induces apoptosis in iCCA cells through ROS/JNK/mitochondrial signaling pathways, and hence, making MLN2238 a potential therapeutic choice for iCCA.

ANLN promotes carcinogenesis in oral cancer by regulating the PI3K/mTOR signaling pathway

BackgroundOral cancer is a malignant disease that threatenshuman life and greatly reducespatientquality of life. ANLN was reported to promote the progression of cancer. This study aims to investigate the role of ANLNin oral cancer and the underlying molecular mechanism.MethodsANLN expression was downregulated by RNAi technology. The effect of ANLN on cell behaviors, including proliferation, cell cycle progression, invasion, and apoptosis, was detected. Western blotting analysis was used to explore the mechanism by whichANLN functions in oral cancer.ResultsData from TCGA database showed that ANLN was expressed at significantly higher levels in tumor tissues thanin normal control tissues. Patients with higher ANLN expression exhibitedshorter survivaltimes. ANLN was alsoabundantly expressedin the cancer cell lines CAL27 and HN30. When ANLN was knocked down in CAL27 and HN30 cells, cell proliferation and colony formation weredecreased. The cell invasion ability was also inhibited. However, the cell apoptosis rate was increased. In addition, the levels of critical members of the PI3K signaling pathway, includingPI3K, mTOR, Akt, and PDK-1, were significantlyreducedafter ANLN was knocked down in CAL27 cells.ConclusionsANLN contributes to oral cancerprogressionand affects activation ofthe PI3K/mTOR signaling pathway. This study providesa new potential targetfor drug development and treatment in oral cancer.

Association between lincRNA expression and overall survival for patients with triple-negative breast cancer.

Long intergenic non-coding RNAs (lincRNAs) are increasingly recognized as important regulators for pathogenesis and/or prognosis of breast cancer, including triple-negative breast cancer (TNBC) subtype. However, few previous studies used RNA-sequencing (RNA-Seq) technology, and none included an independent replication. To systematically evaluate the association between expression of lincRNAs and TNBC survival, we examined lincRNA expression profiles in TNBC tissues using RNA-Seq data for 200 TNBC patients from the Shanghai Breast Cancer Survival Study (SBCSS) and Southern Community Cohort Study (SCCS). Twenty-five lincRNAs were found to be associated with overall survival (P < 0.05 and no significant heterogeneity across studies at Q statistic P > 0.1), and 61 lincRNAs were associated with disease-free survival (DFS). Among these, two lincRNAs (LINC01270 and LINC00449) were significantly associated with both worse overall survival and DFS and were expressed at significantly higher levels in tumor tissues compared with adjacent normal breast tissues (log2[Fold Change] > 0.5 and FDR < 0.05). We further evaluated the potential functions of LINC01270 and LINC00449 using in vitro functional experiments and found that siRNA-mediated knockdown of LINC01270 and LINC00449 expression significantly decreased cell viability, colony formation and cell migration ability in TNBC cells (P < 0.05). Evidence from observational studies and in vitro experiments indicates that LINC00449 and LINC01270 may be prognostic biomarkers for TNBC.

Transducin-Like Enhancer of Split-1 Inhibits Malignant Behaviors in vitro and Predicts a Better Prognosis in Pancreatic Ductal Adenocarcinoma.

Background: Transducin-like enhancer of split-1 (TLE1), a member of the Groucho/TLE family of transcriptional corepressors, has been reported to be involved in the tumorigenesis of various cancers and function as a clinical prognostic indicator. However, the mechanisms and prognostic significance of TLE1 in pancreatic ductal adenocarcinoma (PDAC) have not been elucidated.Methods: In this study, western blot analyses and real-time polymerase chain reaction (RT-PCR) were employed to evaluate the expression of TLE1 and related proteins in PDAC cell lines. Wound healing, transwell migration and invasion, and Cell Counting Kit-8 (CCK-8) assays were used to determine cell line-specific differences in metastasis and proliferation. Flow cytometry was performed for cell cycle detection. RNA sequencing and bioinformatics were undertaken to explore the molecular mechanisms and potential targeted molecules of TLE1. TLE1 expression in tumor and para-tumor tissues was evaluated by tissue microarray-based immunohistochemistry using a semiquantitative method (H-score) in 262 patients with radical PDAC resection. Correlation, Kaplan–Meier survival, univariate, and multivariate analyses were also performed.Results: Our findings showed that TLE1 expression was common in PDAC cell lines. Upregulation of TLE1 inhibited PDAC cell migration, invasion, and proliferation in vitro by delaying the G0/G1 transition. Immunohistochemistry revealed that TLE1 was specifically expressed in the nucleus and at higher levels in tumor tissues compared with para-tumor tissues. Generally, high TLE1 expression was associated with no vascular invasion. In univariate analyses, high TLE1 expression was associated with longer disease-specific survival (DSS) in all patients and in 16 patient subgroups. In multivariate analyses, TLE1 expression was independently associated with DSS in all patients and four patient subgroups.Conclusion: In conclusion, these results suggest that TLE1 has an inhibitory role in PDAC progression and is a favorable prognostic indicator for patients with resectable PDAC.

Human gene encoding hyaluronan binding protein 1 (HABP1/p32/gC1qR): involvement in signaling cascade

Multiligand binding, multifunctional protein, hyaluronan-binding protein 1 (HABP1/SF2-p32/gC1qR) purified and characterized by our laboratory, is revealed to be present in different cellular compartments under various physiological conditions. While unearthing the functions of HABP1, its regulatory involvement in several cellular processes like cellular growth, cell cycle regulation, host-pathogen interaction, apoptosis induction and autophagic vacuole formation has been reported. The role of this protein in regulation of tumorigenesis has been validated as it promotes cellular migration in melanoma cells, shows increased tumor potency with its overexpression in HepG2 cell line and is present in higher levels in tumor tissues. Here, we have tried to explain the probable mechanism of its involvement in tumor pathogenesis. The fact that HABP1 co-purifies with the RNA splicing factor SF2/ASF/SRSF1 is taken into consideration along with the reports of SF2 being an oncogene, which indicates a possible involvement of HABP1 in splicing reactions having an impact on cellular proliferation and tumorigenesis.

Outcome Disparities in African American Compared with European American Women with ER+HER2- Tumors Treated within an Equal-Access Health Care System.

Breast cancer mortality rates are higher for African American women (AAW) than for any other ethnic group in the United States. Recent reports suggest that outcome disparities between AAW and European American women (EAW) are present in the ER+HER2- subtype. To improve our understanding, pathological characteristics, mortality and molecular profiles from women treated within an equal-access health care system were evaluated. All AAW (n=90) and EAW (n=308) with ER+HER2- tumors were identified. Gene expression profiles were generated from primary breast tumors from 57 AAW and 181 EAW. Pathological characteristics, survival and gene expression analysis were evaluated using chi-square analysis, log-rank tests and ANOVA. Tumors from AAW were significantly more likely to be PR-, Ki67+ and of higher grade. Tumor stage, size and lymph node status did not differ significantly, nor did mortality rates (P=.879). At the molecular level, genes PSPHL and CRYBB2P1 were expressed at significantly higher levels in tumor tissues as well as normal stroma and blood from AAW. Polymorphisms controlling expression of each gene were identified with minor allele frequencies differing significantly between populations but not between cases and controls within each population. Survival disparities were not detected in patients with ER+HER2- tumors treated within an equal-access health care system and molecular differences in tumors were not causal. Thus, outcome disparities in AAW with ER+HER2- tumors are largely attributable to socioeconomic factors affecting access to screening and treatment, rather than reflecting underlying biological differences.

RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma, Cirrhotic and Non-Cirrhotic Liver, and Correlation with Liver Cancer in Brazilian Patients.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Most cases of HCC are associated with cirrhosis related to chronic hepatitis B virus or hepatitis C virus infections. Hypermethylation of promoter regions is the main epigenetic mechanism of gene silencing and has been involved in HCC development. The aim of this study was to determine whether aberrant methylation of RASSF1A and DOK1 gene promoters is associated with the progression of liver disease in Brazilian patients. Methylation levels were measured by pyrosequencing in 41 (20 HCC, 9 cirrhotic, and 12 non-cirrhotic) liver tissue samples. Mean rates of methylation in RASSF1A and DOK1 were 16.2% and 12.0% in non-cirrhotic, 26.1% and 19.6% in cirrhotic, and 59.1% and 56.0% in HCC tissues, respectively, showing a gradual increase according to the progression of the disease, with significantly higher levels in tumor tissues. In addition, hypermethylation of RASSF1A and DOK1 was found in the vast majority (88%) of the HCC cases. Interestingly, DOK1 methylation levels in HCC samples were significantly higher in the group of younger (<40 years) patients, and higher in moderately differentiated than in poorly differentiated tumors (p < 0.05). Our results reinforce the hypothesis that hypermethylation of RASSF1A and DOK1 contributes to hepatocarcinogenesis and is associated to clinicopathological characteristics. RASSF1A and DOK1 promoter hypermethylation may be a valuable biomarker for early diagnosis of HCC and a potential molecular target for epigenetic-based therapy.

Comparison of folate derivate levels in mucosa and tumor tissue in the course of antifolate treatment with pemetrexed.

10612 Background: THF (tetrahydrofolate), methyl-THF and methylene-THF are cornerstones for viability of mucosa with different functions in the cell cycle. Understanding the tumor growth and the role of reduced folates is essential in the understanding of mechanisms that occur during chemotherapy with pemetrexed and folate supplementation. Methods: 37 patients (pts) with operable nonmetastatic rectal adenocarcinoma were enrolled. Pemetrexed 500 mg/m2 was administered every 3 weeks, during 3 cycles with standard comedication (vitamin B12, folic acid, and dexamethasone). Biopsies of tumor and adjacent mucosa were taken before the patient received vitamin supplementation, after supplementation but before antifolate therapy and at surgery. A LC-MS/MS method was used for the determination of THF, methylene-THF and methyl-THF levels. Results: See Table. All levels of the three folate derivates increased significantly after the start of the supplementation, in mucosa as well as in tumor tissue. Mean THF and methylene-THF levels were significantly higher in tumor compared with mucosa at baseline, after folate supplementation and at surgery after 3 cycles of pemetrexed. Conclusions: In a population of 37 pts with rectal cancer, there was a strong and consistent significant difference observed for reduced folates between tumor and mucosa. High tumor tissue levels of reduced folates need further exploration to better understand the biological basis to optimize the treatment in rectal cancer and in other tumor entities. Baseline After supplementation At surgery THF a 463/584 620/901 704/906 b 123 (294) 292 (408) 202 (459) c p = 0.006 p ≤ 0.001 p = 0.012 d 36/37 35/35 37/37 Methyl-THF a 352/436 640/903 639/1065 b 84 (227) 263 (352) 426 (585) c p = 0.051 p ≤ 0.001 p ≤ 0.001 d 37/37 36/36 37/37 Methylene-THF a 830/1,018 1,405/2,067 1,302/1,871 b 188 (718) 662 (1,272) 568 (1,352) c p = 0.018 p ≤ 0.001 p = 0.004 d 37/37 36/36 37/37 a = mean levels mucosa/tumor (M/T) (pmol/g); b = Difference T/M, mean (SD); c = p value*; d= number of pts, M/T. * Wilcoxon signed rank test was used for pairwise comparison T-M, testing whether mean difference is zero. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Lilly

Effective conversion of irinotecan to SN-38 after intratumoral drug delivery to an intracranial murine glioma model in vivo

Irinotecan (CPT-11), a topoisomerase I inhibitor, is a cytotoxic agent with activity against malignant gliomas and other tumors. After systemic delivery, CPT-11 is converted to its active metabolite, SN-38, which displays significantly higher cytotoxic potency. However, the achievement of therapeutically effective plasma levels of CPT-11 and SN-38 is seriously complicated by variables that affect drug metabolism in the liver. Thus the capacity of CPT-11 to be converted to the active SN38 intratumorally in gliomas was addressed. For in vitro studies, 2 glioma cell lines, U87 and U251, were tested to determine the cytotoxic effects of CPT-11 and SN-38 in a dose-dependent manner. In vivo studies were performed by implanting U87 intracranially into athymic/nude mice. For a period of 2 weeks, SN-38, CPT-11, or vehicle was administered intratumorally by means of an osmotic minipump. One series of experiments measured the presence of SN-38 or CPT-11 in the tumor and surrounding brain tissues after 2 weeks' exposure to the drug. In a second series of experiments, after 2 weeks' exposure to the drug, the animals were maintained, in the absence of drug, until death. The survival curves were then calculated. The results show that the animals that had CPT-11 delivered intratumorally by the minipump expressed SN-38 in vivo. Furthermore, both CPT-11 and SN-38 accumulated at higher levels in tumor tissues compared with uninvolved brain. Intratumoral delivery of CPT-11 or SN-38 extended the average survival time of tumor-bearing animals from 22 days to 46 and 65 days, respectively. These results demonstrate that intratumorally administered CPT-11 can be effectively converted to SN-38 and this method of drug delivery is effective in extending the survival time of animals bearing malignant gliomas.

Tumor tissue levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and outcome following adjuvant chemotherapy in premenopausal lymph node-positive breast cancer patients: A retrospective study

BackgroundWe have previously demonstrated that high tumor tissue levels of TIMP-1 are associated with no or limited clinical benefit from chemotherapy with CMF and anthracyclines in metastatic breast cancer patients. Here, we extend our investigations to the adjuvant setting studying outcome after adjuvant chemotherapy in premenopausal lymph node-positive patients. We hypothesize that TIMP-1 high tumors are less sensitive to chemotherapy and accordingly that high tumor tissue levels are associated with shorter survival.MethodsFrom our original retrospectively collected tumor samples we selected a group of 525 pre-menopausal lymph node-positive patients (adjuvant treatment: CMF, 324 patients; anthracycline-based, 99 patients; no adjuvant chemotherapy, 102 patients). TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. TIMP-1 was analyzed as a continuous variable and as a dichotomized one using the median TIMP-1 concentration as a cut point between high and low TIMP-1 groups. We analyzed the benefit of adjuvant CMF and anthracyclines in univariate and multivariable survival models; endpoints were disease-free (DFS) and overall survival (OS).ResultsIn this selected cohort of high-risk patients, and in the subgroup of patients receiving no adjuvant therapy, TIMP-1 was not associated with prognosis. In the subgroup of patients treated with anthracyclines, when analyzed as a continuous variable we observed a tendency for increasing TIMP-1 levels to be associated with shorter DFS (multivariable analysis, HR 1.75, 95% CI 1.00-3.07, P = 0.05) and a significant association between increasing TIMP-1 and shorter OS in both univariate (HR 3.52, 95% CI 1.54-8.06, P = 0.003) and multivariable analyses (HR 4.19, 95% CI 1.67-10.51, P = 0.002). No statistically significant association between TIMP-1 and DFS was observed in the CMF-treated patients although high TIMP-1 was associated with shorter OS when analyzed as a dichotomized variable (HR 1.64, 95% CI 1.02-2.65, P = 0.04).ConclusionIn the subgroup of patients receiving adjuvant chemotherapy we found an association between shorter survival after treatment in TIMP-1 high patients compared with TIMP-1 low patients, especially in patients receiving anthracycline-based therapy. This suggests that high tumor tissue levels of TIMP-1 might be associated with reduced benefit from classical adjuvant chemotherapy. Our findings should be validated in larger prospective studies.

Tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) and survival following adjuvant chemotherapy in pre-menopausal lymph node-positive breast cancer patients (N=525).

Abstract Abstract #6054 Predictive markers are needed to guide planning of adjuvant therapy for breast cancer patients. We have recently shown that high tumor tissue levels of TIMP-1 are associated with decreased response to chemotherapy in metastatic breast cancer (Schrohl et al, Clin Cancer Res, 2006) suggesting that TIMP-1 may be a predictive marker in breast cancer patients.&amp;#x2028; Purpose: This study investigates the association of tumor tissue TIMP-1 levels with response to adjuvant chemotherapy with CMF (cyclophosphamide/methotrexate/5-fluorouracil) or an anthracycline-based regimen.&amp;#x2028; Patients and Methods: 525 pre-menopausal lymph node-positive patients were included; 324 patients received adjuvant CMF, 99 received an adjuvant anthracycline-containing regimen and 102 had no adjuvant chemotherapy. Total TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. Using the untreated patient group as a reference group we analyzed the benefit of adjuvant CMF and anthracyclines in TIMP-1 high and low patients, respectively. The median TIMP-1 concentration was used to dichotomize patients into high and low TIMP-1 groups. End points were disease-free and overall survival (DFS, OS).&amp;#x2028; Results: The median TIMP-1 level in the total patient group was 12,54 ng/mg of total protein (range, 0 – 112,9 ng/mg). TIMP-1 levels in subgroups according to adjuvant therapy were not significantly different (P=0,20). In a multivariate model including basic clinico-pathological parameters, TIMP-1 low and high patients benefited differentially from adjuvant CMF and anthracyclines when compared to untreated patients. In particular, patients with high tumor levels of TIMP-1 had little benefit from adjuvant anthracyclines. Hazard ratios (HR) and 95% confidence intervals (CI) are given in the table for the analysis of DFS. A similar pattern was seen in the analyses of OS. In the CMF-treated group, both TIMP-1 low and high patients had significantly better survival than untreated patients (P&amp;lt;0,01). Among anthracycline-treated patients those with TIMP-1 low tumors appeared to benefit more from the adjuvant therapy than TIMP-1 high patients although these results did not reach significance in the present analyses.&amp;#x2028; &amp;#x2028; Conclusion: This study suggests that high tumor tissue TIMP-1 levels are associated with decreased benefit from adjuvant chemotherapy. Especially in the group treated with anthracycline-based therapy there is a strong tendency for TIMP-1 high tumors to be less sensitive to the treatment. This group, however, is small and should be enlarged to confirm our results. In the group treated with CMF all patients benefit from the therapy with TIMP-1 high patients having slightly less benefit than TIMP-1 low patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6054.

Validation of the predictive value of tissue inhibitor of metalloproteinases-1 for the response to first-line chemotherapy in metastatic breast cancer.

Abstract Abstract #6057 *Würtz SØ and Klintman M contributed equally, Malström P and Brünner N shares the senior authorship.&amp;#x2028; Background. Predictive markers are scarcely represented in breast cancer. TIMP-1 has in a previous study (n = 173) performed in our laboratory been shown to carry predictive information for the response to chemotherapy in metastatic breast cancer as high tumor tissue levels of TIMP-1 were significantly associated with a low objective response rate to treatment with the most frequently used chemotherapeutic drugs (CMF or anthracyclines). The purpose of the present study was to validate these previous results with a new independent patient population.&amp;#x2028; Methods. The TIMP-1 level was measured using a validated ELISA in 168 primary tumor extracts from patients with metastatic breast cancer and levels were associated with the objective response to CMF and anthracyclines. Patients were included in the study provided that they had received chemotherapy for their metastatic disease and that frozen tumor tissue as well as data on their objective response to chemotherapy was available.&amp;#x2028; Results. The median TIMP-1 level in responders was 17.3 (2.9 – 75.8) ng TIMP-1/mg protein and in non-responders it was 19.6 (0-190.3) ng TIMP-1/mg protein. When analysed as a continuous log-transformed variable, increasing tumor levels of TIMP-1 were associated with a decreasing probability of objective response to CMF or anthracyclines (OR = 1.59, 95% CI: 0.97 – 2.62, p = 0.07). This is very similar to the original study and thus supporting previous data. Next, we used a more clinically relevant approach for analysing the data from the validation study. For this analysis, objective response was scored as complete or partial response versus stable disease lasting at least six months (clinical benefit) versus progressive disease (non-response). This analysis showed that increasing levels of TIMP-1 were associated with a poor clinical benefit rate from chemotherapy (OR = 1.56, 95% CI: 0.98 - 2.51, p=0.06). The original study has not previously been analysed using this endpoint. When performing this analysis, similar results were found (OR = 1.62, 95% CI: 1.06 - 2.48, p = 0.02). Combining the new population and the original population in a pooled analysis (n = 341) using a random effects model showed that high levels of TIMP-1 were highly significantly associated with a poor clinical benefit rate from chemotherapy compared with patients with low levels (OR = 1.59, 95% CI: 1.16 - 2.18, p=0.003).&amp;#x2028; Conclusion. The present validation study supports previous findings that primary tumor tissue levels of TIMP-1 carries predictive information in metastatic breast cancer treated with chemotherapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6057.

Tissue inhibitor of metalloproteinases-1 protects the human breast carcinoma cell line MCF-7 S1 against anthracycline-induced cell death by activation of the akt survival pathway.

Abstract Abstract #2019 Würtz S and Schrohl A contributed equally.&amp;#x2028; Background. A large proportion of breast cancer patients are offered chemotherapy following primary surgery, however, many of these patients do not benefit from the treatment. Currently, only few markers exist to predict whether a breast cancer patient will respond to chemotherapy in general or to specific types of chemotherapy. Thus, additional predictive markers need to be identified to ensure a more effective treatment of the patients. In this regard, recent clinical studies performed in our laboratory have demonstrated that high tumor tissue levels of TIMP-1 are associated with a poor response to the most frequently used chemotherapeutic drugs (CMF or anthracyclines) (Schrohl et al., Clin. Cancer Res. 2006 and Klintman et al. manus. in prep.). The purpose of the present study was to investigate the molecular explanation for the clinical findings concerning the predictive value of TIMP-1 using an in vitro approach.&amp;#x2028; Methods. We stably transfected the human breast carcinoma cell line MCF-7 S1 with the human TIMP-1 gene and established single cell clones expressing different levels of TIMP-1. We then compared the sensitivity of these cells to epirubicin and taxol using cell death and apoptosis assay. In addition, we investigated the signalling pathway that could be involved in the TIMP-1 mediated protection against treatment using western blotting of key molecules.&amp;#x2028; Results. We found that clones expressing high levels of TIMP-1 were significantly less sensitive to epirubicin. In contrast, no difference in sensitivity to taxol was demonstrated between high and low TIMP-1 clones. When investigating the signalling pathway involved in the difference in sensitivity we found that phosphorylated Akt was highly up-regulated in the high TIMP-1 clones compared to the low TIMP-1 clones following chemotherapeutic treatment.&amp;#x2028; Conclusion. TIMP-1 protects the MCF-7 S1 cells against anthracycline-induced cell death but not against taxol. Thus, TIMP-1 may be used to discriminate between patients likely to benefit from anthracyclines and patients who should be offered an alternative drug. Furthermore, we found that this protection is probably executed through counteraction of the apoptotic signal by activation of the Akt survival pathway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2019.

Platinum Resistance Related to a Functional NER Pathway

Tobacco carcinogens induce DNA adducts that are repaired by the nucleotide excision repair (NER) pathway.1 Inhaled combustion-derived particles, such as cigarette smoke, cause a local pulmonary inflammatory response that is characterized by the influx of neutrophils into the airways. On entering the lung, neutrophils are activated and release reactive oxygen species and an array of proteins, such as myeloperoxidase. A significant reduction of NER in human alveolar epithelial cells was observed when they were cocultured with activated neutrophils.2 NER, a highly versatile pathway for DNA damage removal, is often dysfunctional in non-small cell lung cancer (NSCLC) and could, therefore, be the Achilles heel for customizing chemotherapy. NER removes numerous types of DNA helix-distorting lesions, including cisplatinand ultraviolet-induced photo products.1 Inherited defects in the NER process cause serious repair disorders: xeroderma pigmentosum (XP), with extreme risk of ultraviolet-induced skin cancer, and Cockayne syndrome. NER functions by a “cutand-paste” mechanism in which cisplatin damage recognition, local opening of the DNA helix around the lesion, damage excision, and gap filling occur in successive steps1,3–5 (Figure 1). NER is composed of two subpathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which share the same core mechanism but differ in the way lesions are recognized.6 NER-defective XP is classified into seven complementation groups, XPA to XPG. XPC and XPE are specifically defective in GG-NER, which repairs the damage on the nontranscribed strand, whereas the other XP groups involve deficiencies in both TC-NER and GG-NER.7 The first step in GG-NER is damage recognition by the heterodimer XPC/hHR23B, which binds with higher affinity to helix-distorting DNA lesions than to nondamaged, doublestranded DNA1 (Figure 1A). Various NER factors, including transcription factor IIH (TFIIH, a general transcription factor for RNA polymerase II), XPA, replication protein A (RPA), and XPG work together to repair DNA damage (Figure 1B). TFIIH can be divided into two subcomplexes: the core TFIIH (composed of XPB, p62, p52, p44, p34, and p8) and a cdk-activating kinase subcomplex (containing cdk7, cyclin H, and MAT1).7 Both subcomplexes are bridged by XPD. In NER, TFIIH unwinds the duplex DNA around the lesion to allow the recruitment of the NER factors XPA, RPA, XPG, and excision repair cross-complementing 1 (ERCC1)/XPF (Figure 1B). TFIIH, with the two helicases XPB and XPD, opens an approximately 30-base-long DNA segment around the platinum damage. This open intermediate is stabilized by RPA and XPA (Figure 1C). The DNA strand that contains the damaged base(s) is excised by the two NER endonucleases XPG and ERCC1/XPF (Figure 1D). XPG cleaves the damaged DNA strand 3= from the lesion, and ERCC1/XPF cleaves the damaged strand 5= from the lesion (Figure 1D). The resulting gap is filled by DNA polymerase or in the presence of replication factors8 (Figure 1E). Importantly, the ERCC1/XPF structure-specific nuclease has an additional role in the repair of cisplatin adducts besides its function in NER: the recombination repair of interstrand cross-links.9 Moreover, colocalization of ERCC1 foci and RAD51 foci in response to cisplatin treatment has recently been found and may represent recruitment of ERCC1/XPF to sites of recombination repair.10 Nevertheless, in addition to NER, cisplatininduced cytotoxicity requires the interaction of components from several different DNA damage-processing systems.11

High serum TIMP-1 correlates with poor prognosis in breast carcinoma – A validation study

A number of studies have demonstrated that high tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) are associated with a poor prognosis in breast cancer, suggesting that TIMP-1 could be a valid prognostic marker in this disease. Recently, our laboratories have presented results showing that TIMP-1 also carries prognostic information when measured in serum. This is an important finding, since serum is a much more preferable material compared with tumor tissue extracts. The aim of the present study was to validate the previous results concerning the prognostic value of TIMP-1 in serum obtained preoperatively from 68 patients with primary breast cancer. This was done by measuring the same serum samples as in the previous study but in a different laboratory using a different ELISA assay. We confirmed that patients with the highest serum levels of TIMP-1 (> 197.7 ng/ml) had significantly shorter disease-specific survival compared with patients with low serum TIMP-1 levels. In the group of node-negative patients, 53% of the patients with high levels of TIMP-1 survived after 10 years of follow-up compared to 92% of the patients with low levels. This study thus confirms the reproducibility across laboratories of the results concerning the prognostic value of TIMP-1 in serum. We also investigated whether measurements of the specific fraction of uncomplexed TIMP-1 improved the prognostic value of TIMP-1 in serum, as has been shown to be the case for tumor tissue extracts. However, including information of the level of uncomplexed TIMP-1 did not seem to provide additional prognostic information to that already provided by total TIMP-1.

M06-01: The predictive and prognostic roles of ERCC1 Expression and Akin DNA repair genes in non-small-cell lung cancer

M06-01: The predictive and prognostic roles of ERCC1 Expression and Akin DNA repair genes in non-small-cell lung cancer

Molecular Basis for the Inhibition of Anticancer Agents-induced Apoptosis by Thymidine Phosphorylase

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. TP was expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. Jurkat cells transfected with TP cDNA (Jurkat/TP) and mutant TP cDNA (Jurkat/TPMu) expressed high levels of TP, while Jurkat/CV cells which were transfected with a control vector did not express TP. A high TP enzyme activity was detected in Jurkat/TP cells, but not in Jurkat/CV and Jurkat/TPMu cells. Sensitivities to cisplatin of these cells were determined by MTT assay. IC50 values for cisplatin of Jurkat/CV, Jurkat/TP, and Jurkat/TPMu cells were 4.50, 14.08, 13.40 microM, respectively. Jurkat/TP and Jurkat/TPMu cells were about three times more resistant to cisplatin than Jurkat/CV cells. TP inhibited activation of caspase 3, 9 and mitochondrial cytochrome c release induced by cisplatin. These findings suggest a mechanism by which TP confers the resistance to cisplatin-induced apoptosis. Moreover, mutant TP that has no enzymatic activity also suppressed the cisplatin-induced apoptosis. These suggest that TP molecules have cytoprotective functions against cytotoxic agents.