Molecular Oncology - Articles in Press

ESR1 gene status correlates with estrogen receptor protein levels measured by ligand binding assay and immunohistochemistry - Uncorrected Proof

2012-05-14

Abstract: The Estrogen Receptor (ER) is an established predictive marker for the selection of adjuvant endocrine treatment in early breast cancer. During the 1990s Immunohistochemistry (IHC) replaced cytosol based assays for determination of ER status. This study examined the association between ER protein level determined by two different methods and ESR1 gene copy number. From 289 primary high-risk breast cancer patients, randomized in the Danish Breast Cancer Cooperative Group (DBCG) 77C trial, results from cytosolic ER levels were available from ligand binding assays. Archival tumor tissue was retrieved from 257 patients. ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH) in 220 (86%) patients. ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the patients and ESR1 deletion (ESR1/CEN-6 < 0.80) was observed in 32%. Further, we identified ESR1 gain (ratio ESR1/CEN-6 from 1.30 to 1.99) in 19% of the patients. A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001). Amplification and gain of the ESR1 gene are associated with higher ER protein content measured by ligand binding assay and a more intense nuclear staining by IHC compared to tumors with normal ESR1 gene status. Major variations in ER measured by ligand binding assay and IHC are observed within all ESR1 copy number subgroups and other mechanisms than gene copy number seem to contribute to the ER protein content in the tumors.Highlights: ► The Estrogen Receptor (ER) is an established predictive marker for adjuvant endocrine treatment in early breast cancer. ► We examined the association between ER protein level determined by two different methods and ESR1 gene copy number. ► ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH). ► ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the cases. ► A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001).

Co-administration phenoxodiol with doxorubicin synergistically inhibit the activity of sphingosine kinase-1 (SphK1), a potential oncogene of osteosarcoma, to suppress osteosarcoma cell growth both in vivo and in vitro - Corrected Proof

2012-05-10

Abstract: Elucidation of the mechanisms of chemo-resistance and implementation of strategies to overcome it will be pivotal to improve the survival for osteosarcoma (OS) patients. We here suggest that sphingosine kinase-1 (SphK1) might be the key factor contributing to chemo-resistance in OS. Our Western-blots and immunohistochemistry results showed that SphK1 is over-expressed in multiple clinical OS tissues. Over-expression of SphK1 in OS cell line U2OS promoted its growth and endorsed its resistance against doxorubicin, while knocking-down of SphK1 by shRNA inhibited U2OS cell growth and increased its sensitivity to doxorubicin. Co-administration phenoxodiol with doxorubicin synergistically inhibited SphK1 activity to trigger cellular ceramide accumulation, and achieved synergistic anti-OS growth effect, accompanied with a significant increased of apoptosis and cytotoxicity. Increased cellular level of ceramide by the co-administration induced the association between Akt and Protein Phosphatase 1 (PP1) to dephosphorylate Akt, and to introduce a constitutively active Akt (CA-Akt) restored Akt activation and diminished cell growth inhibition. Further, phenoxodiol and doxorubicin synergistically activated apoptosis signal-regulating kinase 1(ASK1)/c-jun-NH2-kinase (JNK) signaling, which also contributed to cell growth inhibition. Significantly, the role of SphK1 in OS cell growth and the synergistic anti-OS effect of phenoxodiol and doxorubicin were also seen in a mice OS xenograft model. In conclusion, our data suggest that SphK1 might be a critical oncogene of OS and co-administration phenoxodiol with doxorubicin synergistically inhibited the activity of SphK1 to suppress osteosarcoma cell growth both in vivo and in vitro.Highlights: ► SphK1 is over-expressed in multiple clinical osteosarcoma (OS) tissues. ► SphK1 promotes OS cell growth and confers resistance against doxorubicin. ► SphK1 inhibition suppresses OS cell growth and increases doxorubicin sensitivity. ► Phenoxodiol and doxorubicin synergistically inhibit SphK1 to increase ceramide. ► Phenoxodiol and doxorubicin synergistically inhibit Akt, while activating ASK1/JNK.

Oncosuppressive role of p53-induced miR-205 in triple negative breast cancer - Corrected Proof

2012-04-26

Abstract: An increasing body of evidence highlights an intriguing interaction between microRNAs and transcriptional factors involved in determining cell fate, including the well known “genome guardian” p53. Here we show that miR-205, oncosuppressive microRNA lost in breast cancer, is directly transactivated by oncosuppressor p53.Moreover, evaluating miR-205 expression in a panel of cell lines belonging to the highly aggressive triple negative breast cancer (TNBC) subtype, which still lacks an effective targeted therapy and characterized by an extremely undifferentiated and mesenchymal phenotype, we demonstrated that this microRNA is critically down-expressed compared to a normal-like cell line. Re-expression of miR-205 where absent strongly reduces cell proliferation, cell cycle progression and clonogenic potential in vitro, and inhibits tumor growth in vivo, and this tumor suppressor activity is at least partially exerted through targeting of E2F1, master regulator of cell cycle progression, and LAMC1, component of extracellular matrix involved in cell adhesion, proliferation and migration.Highlights: ► miR-205 is downregulated in triple negative breast cancer. ► miR-205 directly targets LAMC1 and E2F1. ► miR-205 inhibits cellular proliferation in vitro and in vivo. ► miR-205 induces cellular senescence. ► p53 regulates miR-205 expression.

Identification of recurrence-related microRNAs in hepatocellular carcinoma following liver transplantation - Corrected Proof

2012-04-23

Abstract: Tumor recurrence-related microRNAs (miRNAs) in hepatocellular carcinoma (HCC) following orthotopic liver transplantation (OLT) are not clear yet. This study was designed to determine whether altered miRNA expression is associated with HCC recurrence and prognosis following OLT. 18 miRNAs, including 6 up-regulated and 12 down-regulated miRNAs were identified by microarray in primary HCC samples of patients who had developed HCC recurrence (n = 5) compared to those with non-recurrence (n = 5) following OLT by using p < 0.05 as cutoff value. The six most significantly altered miRNAs (fold change ≥ 2: miR-19a, miR-886-5p, miR-126, miR-223, miR-24 and miR-147) were further confirmed by qRT-PCR in the remaining 105 HCC samples. In receiver-operating characteristic curve analysis, this six miRNAs were of high sensitivity and specificity in predicting HCC recurrence. Using Cox regression and risk score analysis, we built a six-miRNA signature based on their qRT-PCR readings for the prediction of outcome of HCC following OLT. Kaplan–Meier and Cox proportional regression revealed this six-miRNA signature was a significant independent predictor of overall survival (log-rank p = 0.020) and recurrence-free survival (log-rank p < 0.001). Finally, the data were further reconfirmed in an independent cohort of 50 patients from another transplant center. In addition, bioinformatics Gene Ontology and pathway analysis were also performed to better understand the critical roles of these miRNAs in HCC recurrence. Our study, in addition to suggesting a different miRNA expression pattern between HCC samples of patients with recurrence and those with non-recurrence, proposes that this six-miRNA signature may serve as biomarker for prognosis of HCC patients following OLT.Highlights: ► Altered miRNA expression related with HCC recurrence following OLT was identified. ► These miRNAs could accurately predict HCC recurrence following OLT. ► These miRNAs may serve as biomarkers for outcomes of HCC patients following OLT.

Curcumin inhibits tumor proliferation induced by neutrophil elastase through the upregulation of α1-antitrypsin in lung cancer - Corrected Proof

2012-04-09

Abstract: Lung carcinogenesis is a complex process in an unregulated inflammatory environment. Curcumin has been extensively investigated as a multi-target anti-tumor and anti-inflammation compound. In this paper, we demonstrate a novel inflammation-related mechanism for curcumin-induced inhibition of lung tumor growth. We found that neutrophil elastase, an important regulator of inflammatory processes, directly triggered tumor cell proliferation in human lung adenocarcinoma A549 cells, and curcumin could completely suppress the excess tumor proliferation induced by neutrophil elastase. α1-antitrypsin is synthesized by tumor cells and is the natural inhibitor of neutrophil elastase. We found that curcumin counteracted the decrease of α1-antitrypsin induced by neutrophil elastase by inducing the promoter activity of α1-antitrypsin and promoting its expression in A549 cells. The inhibition of neutrophil elastase-induced proliferation by curcumin was dependent on the PI3K/Akt pathway. Knockdown of α1-antitrypsin by siRNA further enhanced the tumor cell proliferation induced by neutrophil elastase and significantly blocked the anti-proliferation effect of curcumin against neutrophil elastase. Curcumin remarkably inhibited the primary tumor growth of Lewis lung carcinoma (LLC) in C57BL/6 mice. We further showed that curcumin upregulated the level of α1-antitrypsin in primary tumor tissue by promoting its local expression, and the protein level of neutrophil elastase in tumor tissue was obviously decreased in mice treated with curcumin. Overall, our results suggest that neutrophil elastase and α1-antitrypsin play important roles in modulating lung tumor proliferation in inflammatory microenvironment and curcumin inhibits neutrophil elastase-induced tumor proliferation via upregulating α1-antitrypsin expression in vitro and in vivo.Highlights: ► Neutrophil elastase (NE) directly triggers lung tumor cell proliferation. ► Curcumin completely suppresses the excess tumor proliferation induced by NE. ► Curcumin blocks NE-induced tumor proliferation via upregulating α1-antitrypsin in lung cancer. ► Curcumin is a potential alternative against inflammation-related tumor growth.

Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in basal- and luminal-like breast cancer xenografts - Corrected Proof

2012-04-09

Abstract: Several clinical trials have investigated the efficacy of bevacizumab in breast cancer, and even if growth inhibiting effects have been registered when antiangiogenic treatment is given in combination with chemotherapy no gain in overall survival has been observed. One reason for the lack of overall survival benefit might be that appropriate criteria for selection of patients likely to respond to antiangiogenic therapy in combination with chemotherapy, are not available.To determine factors of importance for antiangiogenic treatment response and/or resistance, two representative human basal- and luminal-like breast cancer xenografts were treated with bevacizumab and doxorubicin alone or in combination. In vivo growth inhibition, microvessel density (MVD) and proliferating tumor vessels (pMVD = proliferative microvessel density) were analysed, while kinase activity was determined using the PamChip Tyrosine kinase microarray system.Results showed that both doxorubicin and bevacizumab inhibited basal-like tumor growth significantly, but with a superior effect when given in combination. In contrast, doxorubicin inhibited luminal-like tumor growth most effectively, and with no additional benefit of adding antiangiogenic therapy. In agreement with the growth inhibition data, vascular characterization verified a more pronounced effect of the antiangiogenic treatment in the basal-like compared to the luminal-like tumors, demonstrating total inhibition of pMVD and a significant reduction in MVD at early time points (three days after treatment) and sustained inhibitory effects until the end of the experiment (day 18). In contrast, luminal-like tumors only showed significant effect on the vasculature at day 10 in the tumors having received both doxorubicin and bevacizumab.Kinase activity profiling in both tumor models demonstrated that the most effective treatment in vivo was accompanied with increased phosphorylation of kinase substrates of growth control and angiogenesis, like EGFR, VEGFR2 and PLCγ1. This may be a result of regulatory feedback mechanisms contributing to treatment resistance, and may suggest response markers of value for the prediction of antiangiogenic treatment efficacy.Highlights: ► Bevacizumab inhibits basal-like better than luminal-like tumor growth, in vivo. ► Bevacizumab inhibits vascular proliferation better in basal-like xenografts. ► Bevacizumab improve chemotherapy treatment efficacy in sensitive tumors. ► Kinase activity results suggest feedback mechanisms when tumors acquire resistance.

ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a γ-secretase inhibitor and irinotecan in colorectal cancer - Corrected Proof

2012-04-05

Abstract: The Notch signaling pathway has been shown to be upregulated in colorectal cancer (CRC) and important for the self-renewal of cancer stem cells. In this study, we evaluated the efficacy of PF-03084014, a γ-secretase inhibitor, in combination with irinotecan to identify the effects of treatment on tumor recurrence and the tumor-initiating population in our CRC preclinical explant model. The combination of PF-03084014 and irinotecan had the greatest effect at reducing tumor growth on four CRC tumors when compared with treatment with PF-03084014 or irinotecan alone. The combination significantly reduced tumor recurrence in two CRC explants (CRC001 and CRC036) after treatment was discontinued. Both of these tumors exhibited elevated baseline levels of Notch pathway activation as well as an increase in NOTCH1 gene copy number when compared with the two CRC explants (CRC026 and CRC027) where tumors reappeared quickly after termination of treatment. Isolation and injection of aldehyde dehydrogenase (ALDH+ and ALDH−) cells in an in vivo explant model demonstrated that the ALDH+ cell population were tumorigenic. Evaluation of the ALDH+ cells after 28 days of treatment showed that the combination reduced the ALDH+ population in the tumors that did not regrow. Furthermore, ALDH+ cells from CRC001 and CRC027 were injected in vivo and treated immediately for 28 days. Two months after treatment, tumors were evident in the combination treatment group for CRC027 but not for CRC036. These results indicate the combination of PF-03084014 and irinotecan may be effective in reducing tumor recurrence in CRC patients whose tumors exhibit elevated levels of the Notch pathway.Highlights: ► PF-03084014+irinotecan had the greatest effect at reducing tumor growth. ► Combination slowed tumor recurrence in two CRC explants after treatment was stopped. ► Regrowth sensitive tumors exhibited an increase in NOTCH1 gene copy number. ► The combination reduced the ALDH+ TIC population in regrowth sensitive tumors.

Indirubin derivatives induce apoptosis of chronic myelogenous leukemia cells involving inhibition of Stat5 signaling - Corrected Proof

2012-02-22

Abstract: Indirubin is the major active anti-tumor component of a traditional Chinese herbal medicine used for treatment of chronic myelogenous leukemia (CML). While previous studies indicate that indirubin is a promising therapeutic agent for CML, the molecular mechanism of action of indirubin is not fully understood. We report here that indirubin derivatives (IRDs) potently inhibit Signal Transducer and Activator of Transcription 5 (Stat5) protein in CML cells. Compound E804, which is the most potent in this series of IRDs, blocked Stat5 signaling in human K562 CML cells, imatinib-resistant human KCL-22 CML cells expressing the T315I mutant Bcr-Abl (KCL-22M), and CD34-positive primary CML cells from patients. Autophosphorylation of Src family kinases (SFKs) was strongly inhibited in K562 and KCL-22M cells at 5 μM E804, and in primary CML cells at 10 μM E804, although higher concentrations partially inhibited autophosphorylation of Bcr-Abl. Previous studies indicate that SFKs cooperate with Bcr-Abl to activate downstream Stat5 signaling. Activation of Stat5 was strongly blocked by E804 in CML cells. E804 down-regulated expression of Stat5 target proteins Bcl-xL and Mcl-1, associated with induction of apoptosis. In sum, our findings identify IRDs as potent inhibitors of the SFK/Stat5 signaling pathway downstream of Bcr-Abl, leading to apoptosis of K562, KCL-22M and primary CML cells. IRDs represent a promising structural class for development of new therapeutics for wild type or T315I mutant Bcr-Abl-positive CML patients.Highlights: ► We demonstrate that indirubin derivatives (IRDs) block Stat5 signaling in CML cells. ► Inhibition of activated Stat5 signaling is associated with induction of apoptosis. ► These findings suggest important pharmacological mechanism of action of indirubin. ► IRDs have potential as novel chemotherapeutic agents for treatment of CML patients.

EGFR and myosin II inhibitors cooperate to suppress EGFR-T790M-mutant NSCLC cells - Corrected Proof

Huan-Chih Chiu, Teng-Yuan Chang, Chin-Ting Huang, Yu-Sheng Chao, John T.-A. Hsu — 2012-02-16

Abstract: An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non-small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR-T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and β-actin, in the nucleus of H1975 cells carrying the T790M-mutant EGFR. The interactions of EGFR with MYH9 and β-actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9-β-actin interaction, suggesting that the EGFR interaction with MYH9 and β-actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, β-actin, and EGFR were also impaired by CL-387,785, a kinase inhibitor for EGFR-T790M. Furthermore, CL-387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL-387,785 and blebbistatin enhanced the down-regulation of cyclooxygenase-2 (COX-2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti-cancer effects of CL-387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug-resistant EGFR-T790M.Highlights: ► Physical interactions among EGFR, MYH9, and β-actin were observed in H1975 cells. ► Disrupted cytoskeletal integrity reduced EGFR interactions with MYH9 and β-actin. ► Blebbistatin and CL-387,785 synergistically sensitized H1975 cells.

Heterogeneity among RIP-Tag2 insulinomas allows vascular endothelial growth factor-A independent tumor expansion as revealed by studies in Shb mutant mice: Implications for tumor angiogenesis - Corrected Proof

2012-02-06

Abstract: The Shb adapter protein is a signaling intermediate that operates downstream of vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells. The Shb knockout mouse displays a dysfunctional microvasculature and impaired growth of subcutaneously implanted tumor cells. We decided to investigate tumor growth and angiogenesis in the absence of Shb in an inheritable tumor model, the RIP-Tag2 mouse, which produces insulinomas in a manner highly dependent on de novo angiogenesis. We observed a reduced tumor incidence and burden in both RIP-Tag2 Shb−/− and RIP-Tag2 Shb+/− mice. This correlated with a reduced microvascular density, measured as a percentage of insulinoma area positive for CD31 staining, and altered vascular morphology. However, treatment with a VEGF-A blocking antibody was without effect on the Shb mutant tumor volume whereas it significantly inhibited tumor volume in the wild-type mice, suggesting that in mice with reduced Shb expression tumor angiogenesis was primarily sustained by VEGF-A independent pathway(s). This notion was further substantiated by gene expression analysis of angiogenic markers showing reduced VEGF-A expression in Shb-deficient tumors. Considerable heterogeneity with respect to the gene expression profiles of other angiogenic markers and the signal-transduction characteristics was observed between different tumors, suggesting that multiple “rescue” pathways could be operating. The numbers of invasive tumors or metastases were unchanged in the Shb mutant.It is concluded that the Shb mutant background reduces tumor frequency by chronically suppressing VEGF-A dependent angiogenesis. However, VEGF-A independent angiogenesis supports a significant degree of tumor expansion in Shb-deficient mice, indicating heterogeneity in the mechanisms by which tumor expansion is promoted. Interference with Shb signaling may provide novel means for future cancer therapy.Highlights: ► Shb is an adapter protein operating downstream of VEGFR-2. ► Shb was presently investigated in the context of RIP-Tag2 angiogenesis. ► Absence of Shb reduces RIP-Tag2 tumor burden and VEGF-dependent tumor angiogenesis. ► A fraction of tumors escape the angiogenic inhibition imposed by the absence of Shb. ► Tumor heterogeneity allows escape from angiogenic restriction by various means.

Inhibition of NEDD8-conjugation pathway by novel molecules: Potential approaches to anticancer therapy - Corrected Proof

Tomoaki Tanaka, Tatsuya Nakatani, Tetsu Kamitani — 2012-01-27

Abstract: Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin–proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell-cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8-conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.Highlights: ► We have provided recent insights in the NEDD8-associated researches. ► We have revealed NEDD8-associated molecules regulating the SCF ubiquitin E3 ligases. ► Deneddylation-related proteins are candidates in inhibiting the SCF ligase. ► MLN4924 is a compound involved in initial inhibition of the neddylation cascade.

The toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins - Corrected Proof

2012-01-27

Abstract: Tumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects.This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR.We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells.We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity.Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641.We further demonstrate that SA1641 did not influence the plasma membrane permeability, indicating an intracellular interaction of SA1641 and the toxin components of targeted toxins. Surface plasmon resonance measurements point to a transient binding of SA1641 to the toxin components of targeted toxins.Graphical abstract: Highlights: ► Combination therapy. ► Targeted toxins. ► Efficacy increase by saponins. ► Binding of saponins to toxins.

Structural and genic characterization of stable genomic regions in breast cancer: Relevance to chemotherapy - Corrected Proof

Nicole I. Park, Peter K. Rogan, Heather E. Tarnowski, Joan H.M. Knoll — 2012-01-24

Abstract: Background: Cancer genomes accumulate frequent and diverse chromosomal abnormalities as well as gene mutations but must maintain the ability to survive in vivo. We hypothesize that genetic selection acts to maintain tumour survival by preserving copy number of specific genes and genomic regions. Genomic regions and genes that remain unaltered in copy number and expression, respectively, may be essential for maintaining tumour survival.Methods: We analyzed copy number data of 243 previously reported breast tumours and computationally derived stable copy number regions. To identify genes in stable copy number regions with nominal changes in expression, datasets for tumour and normal samples were compared. Results were replicated by analysis of a series of independent copy number, expression and genomic sequencing studies. A subset of stable regions, including stable paralogous regions, were confirmed by quantitative PCR and fluorescence in situ hybridization (FISH) in 5 breast cancer cell lines. We deduced a comprehensive set of dually stable genes (i.e. maintaining nominal copy number and expression) which were categorized according to pathway and ontology assignments. The stability of genes encoding therapeutic drug targets was also assessed.Results and Conclusion: Tumour genome analysis revealed 766 unstable (amplified and/or deleted) and 812 stable contiguous genomic regions. Replication analysis of an independent set of 171 breast tumours confirmed copy number stability of 1.3 Gb of the genome. We found that 5804 of these genes were dually stable. The composition of this gene set remained essentially unchanged (<2% reduction) after accounting for commonly mutated breast cancer genes found by sequencing and differential expression. The stable breast cancer genome is enriched for cellular metabolism, regulation of gene expression, DNA packaging (chromatin and nucleosome assembly), and regulation of apoptosis functions. Stable genes participating in multiple essential pathways were consistently found to be targets of chemotherapies. Preservation of stable, essential genes may be related to the effectiveness of certain chemotherapeutic agents that act on multiple gene products in this set.Graphical abstract: Highlights: ► A portion of the genome is stable in a large number of breast tumours. ► The breast cancer genome contains stable paralogous gene-rich sequence families. ► Genes with stable copy number and expression are consistent between independent studies. ► Stable genes define essential biochemical pathways/functions in tumours. ► Stable genes encode targets of systemic breast cancer chemotherapies.

Adseverin: A novel cisplatin-resistant marker in the human bladder cancer cell line HT1376 identified by quantitative proteomic analysis - Corrected Proof

2012-01-12

Abstract: Cisplatin is currently the most effective antitumor agent available against bladder cancer. However, a majority of patients eventually relapse with cisplatin-resistant disease. Chemoresistance thus remains a major obstacle in bladder cancer therapy. To clarify the molecular mechanisms underlying cisplatin resistance in bladder cancer, we established a cisplatin-resistant subline from the human bladder cancer cell line HT1376 (HT1376-CisR), and conducted large-scale analyses of the expressed proteins using two-dimensional (2D) gel electrophoresis coupled with mass spectrometry (MS). Comparative proteomic analysis of HT1376 and HT1376-CisR cells revealed 36 differentially expressed proteins, wherein 21 proteins were upregulated and 15 were downregulated in HT1376-CisR cells. Among the differentially regulated proteins, adseverin (SCIN), a calcium-dependent actin-binding protein, was overexpressed (4-fold upregulation) in HT1376-CisR, with the increase being more prominent in the mitochondrial fraction than in the cytosol fraction. SCIN mRNA knockdown significantly reduced cell proliferation with mitochondria-mediated apoptosis in HT1376-CisR cells. Immunoprecipitation analysis revealed voltage-dependent anion channels (VDACs) to be bound to SCIN in the mitochondrial fraction. Our results suggest that the VDAC-SCIN interaction may inhibit mitochondria-mediated apoptosis in cisplatin-resistant cells. Targeting the VDAC-SCIN interaction may offer a new therapeutic strategy for cisplatin-resistant bladder cancer.Highlights: ► We established a cisplatin-resistant subline (HT1376-CisR). ► We used quantitative proteomic analysis to compare between HT1376-CisR and HT1376. ► Adseverin, an actin-binding protein, was overexpressed in HT1376-CisR. ► It affected behaviors of voltage-dependent anion channel, interfering with apoptosis. ► Targeting the VDAC-adseverin interaction may offer a new therapeutic strategy.

The interaction of PKN3 with RhoC promotes malignant growth - Corrected Proof

2012-01-03

Abstract: PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3–RhoC complex represents an attractive therapeutic target in late-stage malignancies.Highlights: ► Inducible PKN3 knockdown inhibits primary prostate and breast tumor growth. ► PKN3 associates with Rho-family GTPases, and preferentially with RhoC. ► RhoC predominantly associates with PKN3 compared to other PKN family members. ► PKN3 catalytic activity is stimulated in the presence of RhoA and RhoC. ► Turn-motif phosphorylation at T860 closely correlates with PKN3 activity.

Serous ovarian carcinoma patients with high alpha-folate receptor had reducing survival and cytotoxic chemo-response - Corrected Proof

2011-12-29

Abstract: The alpha-folate receptor (α-FR) is highly-expressed in various non-mucinous tumors of epithelial origin, including ovarian carcinoma. The aim of this study was to investigate the relationship between alpha-folate receptor (α-FR) and the clinico-pathologic features and outcomes of serous ovarian carcinoma patients and the possible mechanism of α-FR to chemo-resistance. Therefore, semi-quantitative reverse-transcription polymerase chain reactions for α-FR expression were performed in the 91 specimens of serous ovarian carcinomas. The expression of α-FR in each ovarian cancer tissue specimen was defined as the ratio of density of α-FR to density of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In vitro apoptotic experiments were tested in the original OVCAR-3 tumor cells and various OVCAR-3 α-FR-transfectants. Patients with an increased α-FR expression level had poorer responses to chemotherapy (per α-FR expression level increase: odds ratio (OR): 8.97 (95% confidence interval (CI): 1.40–57.36), p = 0.021). An increased α-FR expression level was an independently poor prognostic factor for disease free interval (DFI) (per α-FR expression level increase: hazard ratio (HR): 2.45 (95% CI: 1.16–5.18), p = 0.02) and had a negative impact on overall survival (OS) of these serous ovarian cancer patients (per α-FR expression level increase: HR: 3.6 (95% CI: 0.93–13.29), p = 0.03) by multivariate analyses. α-FR inhibited cytotoxic drug-induced apoptosis in our in vitro apoptotic assays. α-FR could induce chemo-resistance via regulating the expression of apoptosis-related molecules, Bcl-2 and Bax. Therefore, α-FR can be a potential biomarker for the prediction of chemotherapeutic responses and clinical prognosis. It also could be the target of ovarian cancer treatment.Highlights: ► Patients with an increased α-FR expression level had poorer responses to chemotherapy. ► Increased α-FR expression level was a poor prognostic factor for overall survival of the patients. ► Ovarian serous cancer patients with high levels of α-FR had poor chemo-response. ► α-FR can be a biomarker for the outcome of ovarian serous cancer patients.