Table of Contents

Volume 13, Issue 10

  August 2012

  Pages 831 - 966

View covers archive

 Issue Catalogue (MARC XML)

MEETING REPORT

Biomarkers—a pot of gold or a can of worms?: Meeting report from the 2nd World Congress on Biomarkers & Clinical Research, 2011, Baltimore, USA

Georg F. Weber, Jeremy Warren, Hitoshi Shoma, Tao Chen, Abdel Halim and Geetika Chakravarty
Pages 831 - 835
http://dx.doi.org/10.4161/cbt.20847
Abstract | Full Text | PDF

Biomarkers are biological agents used as indicators of biological states. In clinical applications, biomarkers reflect the presence, severity, or progression of disease states. They may also predict risk or responsiveness of a disease to a given treatment. There has been increasingly intense research interest in biomarkers, yet their translation into routine clinical use is lagging. To stimulate communication and cross-fertilization, the 2nd World Congress on Biomarkers & Clinical Research was held in Baltimore, MD, USA in 2011. The symposium covered a broad range of basic and applied biomarker research with the intent to facilitate bench-to-bedside developments. Sessions discussed DNA-based, proteomic, and blood-borne markers. The presentations covered biomarkers for cancer, other various diseases, and toxicological agents. Other topics included biomarker data assimilation, validation, standardization and quality control, as well as molecular imaging and informatics. New high-throughput assays, model systems and emerging technologies give reasons to hope for further rapid progress in the field.




BRIEF COMMUNICATION

Open Access Article

Isocitrate dehydrogenase 1R132H mutation in microglia/macrophages in gliomas: Indication of a significant role of microglia/macrophages in glial tumorigenesis

Ping-Pin Zheng, Marcel van der Weiden, Peter J. van der Spek, Arnaud J.P.E. Vincent and Johan M. Kros
Pages 836 - 839
http://dx.doi.org/10.4161/cbt.20836
Abstract | Full Text | PDF

Somatic mutation of Isocitrate dehydrogenase 1 (IDH1) at the locus of R132 (IDH1^R132H) occurs in > 70% of WHO grade II-III gliomas and secondary glioblastomas. To date it remains unknown whether the mutation is restricted to glial tumor cells. Microglial cells are the resident macrophages in the central nervous system. Tumor-infiltrating microglial cells/macrophages are major stromal cellular components of malignant gliomas and substantially contribute to the tumor mass. Differential identification of the IDH1^R132H mutant cellular components is of particular importance for understanding of the mutation-associated tumor biology. Here we discovered that a significant portion of CD68⁺, Iba1⁺, CX3CR1⁺ microglial cells/macrophages also harbor the IDH1R132H mutation. The findings provide novel insights for understanding the mutation-associated tumor biology relevant to clinical applications as a predictive and/or prognostic marker or therapeutic target.




CLINICAL STUDY

Recurrent gliomas: Comparison of computed tomography (CT)-guided ¹²⁵I seed implantation therapy and traditional radiochemotherapy

Xiaokun Hu, Huijuan Qiu, Liang Zhang, Weidong Zhang, Yongqiang Ma, Zhizheng Qiao, Dong Chen, Jianjun Han, Guangfeng Duan and Fujun Zhang
Pages 840 - 847
http://dx.doi.org/10.4161/cbt.20834
Abstract | Full Text | PDF

Background: Primary brain tumors have always been associated with high morbidity and mortality. Glioma is the most common type of malignant brain tumors,with a high probability of recurrence after surgical excision and with poor prognosis.The purpose of this study was to compare the therapeutic efficacy of computed tomography (CT)-guided interstitial ¹²⁵I seed implantation with traditional radiochemotherapy for treatment of recurrent gliomas.

Results: The response rate at 1, 3, 6 and 12 months after ¹²⁵I seed implantation was 68.6, 74.3, 77.1 and 62.8% respectively, which was significantly higher than the group treated with the conventional chemoradiation protocol (p < 0.05). Patients exposed to ¹²⁵I seed implantation had a median survival of 29.0 months, whereas the median survival of those treated with traditional radiochemotherapy was 19.0 months. The difference observed between the two groups was significant. There were no severe complications or mortality associated with either treatment, except for one case of intracerebral hemorrhage around the tumor area in the ¹²⁵I seed implants group.

Methods: From November 2002 to May 2010, 73 consecutive patients with recurrent gliomas were treated with CT-guided ¹²⁵I seed implantation (35 cases) or traditional radiochemotherapy (38 cases). Patients were followed up after treatment and the therapeutic effect was evaluated by comparing the response and survival rates of the two groups. In particular, patients treated with ¹²⁵I seed implantation were monitored for adverse side effects.

Conclusions: CT-guided ¹²⁵I seed implantation is safe and well-tolerated and more importantly, shows superior efficacy compared with conventional radiochemotherapy. This suggests that CT-guided ¹²⁵I seed implantation could be an alternative approach for recurrent gliomas.




RESEARCH PAPERS

The DNA binding and accumulation of p53 from breast cancer cell lines and the link with serine 15 phosphorylation

Debolina Ray, Keith R. Murphy and Susannah Gal
Pages 848 - 857
http://dx.doi.org/10.4161/cbt.20835
Abstract | Full Text | PDF

Stress treatment generally causes the post-translational modification and accumulation of the p53 protein, although the role of these aspects has not been always understood in relation to this protein’s tumor suppressor activity. We analyzed these attributes of p53 in eight different breast cancer cell lines, with either wild-type or mutant p53 protein, in response to oxidative stress. We found that the wild-type p53 protein from MCF-7 and ZR-75-1 cells binds with different affinity to 12 gene sequences covering several pathways regulated by p53. Treatment of MCF-7 cells with H₂O₂ caused an increase in this binding affinity while this same treatment of ZR-75-1 cells caused the p53 protein to lose binding affinity to several genes. The mutant p53 proteins from all cell lines had minimal to weak binding to these sequences even after treatment with H₂O₂. The p53 protein from the ZR-75-1 cells and three cell lines with mutant p53 showed serine 15 phosphorylated protein, but we found no correlation between that modification and the levels or localization of this protein although DNA binding affinity of wild-type protein might be affected by this modification. From this and other work, it appears that the mutation status of the TP53 gene alone cannot predict the activity of this tumor suppressor since cell lines with the same genetic information do not show the same properties of this protein.

Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

Olga Frolova, Ismael Samudio, Juliana Maria Benito, Rodrigo Jacamo, Steven M. Kornblau, Ana Markovic, Wendy Schober, Hongbo Lu, Yi Hua Qiu, Daniela Buglio, Teresa McQueen, Sherry Pierce, Elizabeth J. Shpall, Sergej Konoplev, Deborah Thomas, Hagop Kantarjian, Richard Lock, Michael Andreeff and Marina Konopleva
Pages 858 - 870
http://dx.doi.org/10.4161/cbt.20838
Abstract | Full Text | PDF

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment.

Exome sequencing and digital PCR analyses reveal novel mutated genes related to the metastasis of pancreatic ductal adenocarcinoma

Bin Zhou, Astrid Irwanto, Yun-Miao Guo, Jin-Xin Bei, Qiao Wu, Ge Chen, Tai-Ping Zhang, Jin-Jv Lei, Qi-Sheng Feng, Li-Zhen Chen, Jianjun Liu and Yu-Pei Zhao
Pages 871 - 879
http://dx.doi.org/10.4161/cbt.20839
Abstract | Full Text | PDF

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers with more than 94% mortality rate mainly due to the widespread metastases. To find out the somatically mutated genes related to the metastasis of PDAC, we analyzed the matched tumor and normal tissue samples from a patient diagnosed with liver metastatic PDAC using intensive exome capture-sequencing analysis (> 170× coverage). Searching for the somatic mutations that drive the clonal expansion of metastasis, we identified 12 genes with higher allele frequencies (AFs) of functional mutations in the metastatic tumor, including known genes KRAS and TP53 for metastasis. Of the 10 candidate genes, 6 (ADRB1, DCLK1, KCNH2, NOP14, SIGLEC1, and ZC3H7A), together with KRAS and TP53, were clustered into a single network (p value = 1 × 10⁻²²) that is related to cancer development. Moreover, these candidate genes showed abnormal expression in PDAC tissues and functional impacts on the migration, proliferation, and colony formation abilities of pancreatic cancer cell lines. Furthermore, through digital PCR analysis, we revealed potential genomic mechanisms for the KRAS and TP53 mutations in the metastatic tumor. Taken together, our study shows the possibility for such personalized genomic profiling to provide new biological insight into the metastasis of PDAC.

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis

Atsushi Kawabata, Takaya Matsuzuka, Chiyo Doi, Garret Seiler, Jennifer Reischman, Lara Pickel, Rie Ayuzawa, Thu A. Nguyen and Masaaki Tamura
Pages 880 - 889
http://dx.doi.org/10.4161/cbt.20840
Abstract | Full Text | PDF

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101–112) and C1B5 (residues 141–151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60–70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32–0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G₂ arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

Suppression of CX43 expression by miR-20a in the progression of human prostate cancer

Xin Li, Jin-Hong Pan, Bo Song, En-Qing Xiong, Zhi-Wen Chen, Zhan-Song Zhou and Yong-Ping Su
Pages 890 - 898
http://dx.doi.org/10.4161/cbt.20841
Abstract | Full Text | PDF

The aberrant expression of microRNAs (miRNAs) has been found in various types of cancer. The present study found miR-20a to be significantly upregulated in prostate cancer compared with normal prostate tissues. The proliferation and colony formation assays revealed that the downregulation of miR-20a by miR-20a inhibitor suppresses the proliferation of MDA-PCa-2b cells in vitro and also inhibits tumor growth in vivo. Furthermore, a gap junction protein, α 1 (CX43), was identified as a direct target gene of miR-20a. The upregulation of CX43 was detected in MDA-PCa-2b cells after treatment with miR-20a inhibitor both in vitro and in vivo. In conclusion, the findings show that miR-20a significantly contributes to the progression of prostate cancer by targeting CX43.

Stromal galectin-1 expression is associated with long-term survival in resectable pancreatic ductal adenocarcinoma

Ru Chen, Sheng Pan, NIki A. Ottenhof, Roeland F. de Wilde, Christopher L. Wolfgang, Zhaoli Lane, Jane Post, Mary P. Bronner, Jürgen K. Willmann, Anirban Maitra and Teresa A. Brentnall
Pages 899 - 907
http://dx.doi.org/10.4161/cbt.20842
Abstract | Full Text | PDF

The overall 5 year survival rate for pancreatic ductal adenocarcinoma (i.e., PDAC) is a dismal 5%, although patients that have undergone surgical resection have a somewhat better survival rate of up to 20%. Very long-term survivors of PDAC (defined as patients with ≥ 10 year survival following apparently curative resection), on the other hand, are considerably less frequent. The molecular characteristics of very long-term survivors (VLTS) are poorly understood, but might provide novel insights into prognostication for this disease. In this study, a panel of five VLTS and stage-matched short-term survivors (STS, defined as disease-specific mortality within 14 months of resection) were identified, and quantitative proteomics was applied to comparatively profile tumor tissues from both cohorts. Differentially expressed proteins were identified in cancers from VLTS vs. STS patients. Specifically, the expression of galectin-1 was 2-fold lower in VLTS compared with STS tumors. Validation studies were performed by immunohistochemistry (IHC) in two additional cohorts of resected PDAC, including: 1) an independent cohort of VLTS and 2) a panel of sporadic PDAC with a considerable range of overall survival following surgery. Immunolabeling analysis confirmed that significantly lower expression of stromal galectin-1 was associated with VLTS (p = 0.02) and also correlated with longer survival in sporadic, surgically-treated PDAC cases (hazard ratio = 4.9, p = 0.002). The results from this study provide new insights to better understand the role of galectin-1 in PDAC survival, and might be useful for rendering prognostic information, and developing more effective therapeutic strategies aimed at improving survival.

Silencing of ribonucleotide reductase subunit M1 potentiates the antitumor activity of gemcitabine in resistant cancer cells

Piyanuch Wonganan, Woon-Gye Chung, Saijie Zhu, Kaoru Kiguchi, John DiGiovanni and Zhengrong Cui
Pages 908 - 914
http://dx.doi.org/10.4161/cbt.20843
Abstract | Full Text | PDF

Gemcitabine is a deoxycytidine analog used for the treatment of a wide range of solid tumors. Its efficacy is however often reduced due to the development of resistance. Ribonucleotide reductase M1 subunit (RRM1) is a key determinant of gemcitabine resistance, and tumor cells that overexpress RRM1 are resistant to the cytotoxicity of gemcitabine. In the present study, we showed that RRM1-specific small interfering RNA (siRNA), when complexed with polyethylenimine, effectively downregulated the expression of RRM1 protein in mouse tumor cells that overexpress RRM1, both in vitro and in vivo. More importantly, systemic administration of the RRM1-specific siRNA significantly inhibited the growth of RRM1-overexpressing tumors in mice and sensitized the tumors to gemcitabine treatment. These findings suggest that silencing RRM1 expression using siRNA could potentially be an effective strategy to overcome gemcitabine resistance.

Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth

YiWei Zhang, Qi Zhang, Shelya X. Zeng, Yu Zhang, Lindsey D. Mayo and Hua Lu
Pages 915 - 924
http://dx.doi.org/10.4161/cbt.20844
Abstract | Full Text | PDF

Several proteins have been suggested in promoting tumor formation in numerous human tissues by inactivating the tumor suppressor p53. This has generated interest in the development of small molecules to block these inhibitors of p53 and to regain p53 activity. Recently, we identified a small molecule, Inauhzin, which can inhibit SIRT1 activity and activate p53. SIRT1 is a deacetylase that deacetylates p53 and facilitates Mdm2 mediated p53 destabilization. In this study, we tested if combining Inauhzin with Nutlin-3, an inhibitor of MDM2-p53 binding, might synergistically activate p53 to suppress tumor growth. Indeed, at lower doses, combination of Inauhzin and Nutlin-3 exhibited a synergistic effect on inhibiting cell growth and promoting apoptosis in human colon and lung cancer cell lines in a p53-dependent fashion. Minimal effects were observed with treatment of either compound alone. Using a xenograft tumor model, we also showed a synergistic effect with both compounds. Thus, to fully regain p53 activity, targeting its multiple inhibitory proteins might be a better approach. Our study provides evidence supporting this concept for achieving better therapeutic efficacy in tumors that possess wild type p53.

Trans-resveratrol boronic acid exhibits enhanced anti-proliferative activity on estrogen-dependent MCF-7 breast cancer cells

Venkata Mahidhar Yenugonda, Yali Kong, Tushar B. Deb, Yonghong Yang, Rebecca B. Riggins and Milton L. Brown
Pages 925 - 934
http://dx.doi.org/10.4161/cbt.20845
Abstract | Full Text | PDF

Resveratrol (RSV), a natural compound present in the skin and seeds of red grapes, is considered a phytoestrogen and has structural similarity to the synthetic estrogen diethylstilbestrol. RSV inhibits tumor cell growth in estrogen receptor-positive (ER+) and negative (ER-) breast cancer cell lines resulting in cell specific regulation of the G₁/S and G₂/M stages of the cell cycle. However apoptotic cell death was only observed in ER+ MCF-7 cells. In this study, we designed and synthesized boronic acid derivative of RSV and evaluated their biological effects on ER+ MCF-7 breast cancer cells. The trans-4 analog inhibited the growth of MCF-7 cells and is not a substrate for p-glycoprotein. The trans-4 analog induces G₁ cell cycle arrest, which coincides with marked inhibition of G₁ cell cycle proteins and a greater pro-apoptotic effect. Finally, the trans-4 analog had no effect on the estrogen-stimulated growth of MCF-7 cells. Our results demonstrate that the trans-4 analog inhibits MCF-7 breast cancer cells by a different mechanism of action than that of RSV (S-phase arrest), and provides a new class of novel boronic acids of RSV that inhibit breast cancer cell growth.

Preclinical modeling of EGFR inhibitor resistance in head and neck cancer

Kelly M. Quesnelle, Sarah E. Wheeler, Mary K. Ratay and Jennifer R. Grandis
Pages 935 - 945
http://dx.doi.org/10.4161/cbt.20846
Abstract | Full Text | PDF

The epidermal growth factor receptor (EGFR) is widely expressed in head and neck squamous cell carcinomas (HNSCC) and can activate many growth and survival pathways within tumor cells. Despite ubiquitous EGFR expression, therapies targeting the receptor are only modestly effective in the treatment of HNSCC. A consistent mechanism of resistance to EGFR targeting agents has not yet been identified in HNSCC likely due, in part, to the paucity of preclinical models. We assessed the in vitro and in vivo responses of a panel of 10 genotypically validated HNSCC cell lines to the EGFR inhibitors erlotinib and cetuximab to determine their validity as models of resistance to these agents. We defined a narrow range of response to erlotinib in HNSCC cells in vitro and found a positive correlation between EGFR protein expression and erlotinib response. We observed cross-sensitivity in one HNSCC cell line, 686LN, between erlotinib and cetuximab in vivo. We attempted to generate models of cetuximab resistance in HNSCC cell line-derived xenografts and heterotopic tumorgrafts generated directly from primary patient tumors. While all 10 HNSCC cell line xenografts tested were sensitive to cetuximab in vivo, heterotopic patient tumorgrafts varied in response to cetuximab indicating that these models may be more representative of clinical responses. These studies demonstrate the limitations of using HNSCC cell lines to reflect the heterogeneous clinical responses to erlotinib and cetuximab, and suggest that different approaches including heterotopic tumorgrafts may prove more valuable to elucidate mechanisms of clinical resistance to EGFR inhibitors in HNSCC.

HuR’s post-transcriptional regulation of death receptor 5 in pancreatic cancer cells

Danielle M. Pineda, David W. Rittenhouse, Christopher C. Valley, Joseph A. Cozzitorto, Richard A. Burkhart, Benjamin Leiby, Jordan M. Winter, Matthew C. Weber, Eric R. Londin, Isidore Rigoutsos, Charles J. Yeo, Myriam Gorospe, Agnieska K. Witkiewicz, Jonathan N. Sachs and Jonathan R. Brody
Pages 946 - 955
http://dx.doi.org/10.4161/cbt.20952
Abstract | Full Text | PDF

Apoptosis is one of the core signaling pathways disrupted in pancreatic ductal adenocarcinoma (PDA). Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF)-receptor superfamily that is expressed in cancer cells. Binding of TNF-related apoptosis-inducing ligand (TRAIL) to DR5 is a potent trigger of the extrinsic apoptotic pathway, and numerous clinical trials are based on DR5-targeted therapies for cancer, including PDA. Human antigen R (HuR), an RNA-binding protein, regulates a select number of transcripts under stress conditions. Here we report that HuR translocates from the nucleus to the cytoplasm of PDA cells upon treatment with a DR5 agonist. High doses of DR5 agonist induce cleavage of both HuR and caspase 8. HuR binds to DR5 mRNA at the 5′-untranslated region (UTR) in PDA cells in response to different cancer-associated stressors and subsequently represses DR5 protein expression; silencing HuR augments DR5 protein production by enabling its translation and thus enhances apoptosis. In PDA specimens (n = 53), negative HuR cytoplasmic expression correlated with elevated DR5 expression (odds ratio 16.1, p < 0.0001). Together, these data demonstrate a feedback mechanism elicited by HuR-mediated repression of the key apoptotic membrane protein DR5.

Bif-1 suppresses breast cancer cell migration by promoting EGFR endocytic degradation

Kristin B. Runkle, Cheryl L. Meyerkord, Neelam V. Desai, Yoshinori Takahashi and Hong-Gang Wang
Pages 956 - 966
http://dx.doi.org/10.4161/cbt.20951
Abstract | Full Text | PDF

Dysregulation of EGFR expression and signaling is well documented to contribute to disease progression and metastasis in many types of cancer including breast cancer. EGF-stimulated EGFR activation leads to receptor internalization and endocytic degradation to control EGFR-mediated signaling. This process is frequently deregulated in cancer cells, leading to increased EGFR expression and mitogenic signaling. Here, we demonstrate that Bif-1, a tumor suppressor, plays a role in EGFR endocytic degradation and chemotactic migration in MDA-MB-231 breast cancer cells. Our data reveal that suppression of Bif-1 expression delays EGFR degradation and sustains Erk1/2 activation in response to EGF stimulation. Mechanistically, loss of Bif-1 sequesters internalized EGF in Rab5-positive endosomes and delays EGFR trafficking to lysosomes. Recruitment of Rab7 to EGF-positive vesicles and the activation of Rab7 are impaired in Bif-1 knockdown cells. Additionally, intracellular pH and the localization of acidic vesicles are altered by suppression of Bif-1. Furthermore, inhibition of Bif-1 increases chemotactic cell migration in response to EGF or serum, which correlates with prolonged cytoskeletal reorganization. Importantly, the effect of Bif-1 on EGF-induced cell migration is abolished by gefitinib, an EGFR-specific inhibitor. Taken together, these data suggest a novel function for Bif-1 as a suppressor of breast cancer cell migration by promoting EGFR degradation through the regulation of endosome maturation.