Ofer Eidelman

Haploinsufficiency of Anx7 tumor suppressor gene and consequent genomic instability promotes tumorigenesis in the Anx7(+/-) mouse

Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7(+/-) knockout mouse. As hypothesized, the Anx7(+/-) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(+/-) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes.

Proteomic biomarkers for blast neurotrauma: targeting cerebral edema, inflammation, and neuronal death cascades.

Proteomics for blast traumatic brain injury (bTBI) research represents an exciting new approach that can greatly help to address the complex pathology of this condition. Antibody-based platforms, antibody microarrays (AbMA), and reverse capture protein microarrays (RCPM) can complement the classical methods based on 2D gel electrophoresis and mass spectrometry (2DGE/MS). These new technologies can address problematic issues, such as sample complexity, sensitivity, quantitation, reproducibility, and analysis time, which are typically associated with 2DGE/MS. Combined with bioinformatics analysis and interpretation of primary microarray data, these methods will generate a new level of understanding about bTBI at the level of systems biology. As biological and clinical knowledge and the availability of these systems become more widely established, we expect that AbMA and RCPM will be used routinely in clinical diagnostics, and also for following therapeutic progress. At the technical level, we anticipate that these platforms will evolve to accommodate comprehensive, high-speed, label-free analysis on a human proteome-wide scale.

Restoration of the cystic fibrosis transmembrane conductance regulator function by splicing modulation

A significant fraction of disease‐causing mutations affects pre‐mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Overexpression of splicing factors increased the level of correctly spliced RNA, transcribed from minigenes carrying disease‐causing splicing mutations. However, whether this increase could restore the protein function was unknown. Here, we demonstrate that overexpression of Htra2‐β1 and SC35 increases the level of normal cystic fibrosis transmembrane conductance regulator (CFTR) transcripts in cystic‐fibrosis‐derived epithelial cells carrying the 3849+10 kb C → T splicing mutation. This led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. These results highlight the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

Curcumin treatment suppresses IKKβ kinase activity of salivary cells of patients with head and neck cancer: a pilot study.

Purpose: To determine whether curcumin would inhibit IκB kinase β (IKKβ) kinase activity and suppress expression of proinflammatory cytokines in head and neck squamous cell carcinoma cancer (HNSCC) patients. Experimental Design: Saliva was collected before and after subjects chewed curcumin tablets. Protein was extracted and IKKβ kinase activity measured. Interleukin (IL)-6 and IL-8 levels in the salivary supernatants were measured by ELISA. IL-6, IL-8, and other interleukin were also measured independently with ELISA to confirm the inhibitory effect of curcumin on expression and secretion of salivary cytokines. Results: Curcumin treatment led to a reduction in IKKβ kinase activity in the salivary cells of HNSCC patients (P < 0.05). Treatment of UM-SCC1 cells with curcumin as well as with post-curcumin salivary supernatant showed a reduction of IKKβ kinase activity. Significant reduction of IL-8 levels (P < 0.05) was seen in post-curcumin samples from patients with dental caries. Although there was reduced IL-8 expression in 8 of 21 post-curcumin samples of HNSCC patients, the data did not reach statistical significance. Saliva samples from HNSCC patients were also analyzed in a blinded fashion for expression of cytokines. IL-10, IFN-γ, IL-12p70, and IL-2 clustered together, and granulocyte macrophage colony stimulating factor and TNF-α clustered together. Log10 ratio analysis showed decrease in expression of all nine cytokines in both the salivary supernatant and salivary cells of curcumin-treated samples. Conclusions: Curcumin inhibited IKKβ kinase activity in the saliva of HNSCC patients, and this inhibition correlated with reduced expression of a number of cytokines. IKKβ kinase could be a useful biomarker for detecting the effect of curcumin in head and neck cancer. Clin Cancer Res; 17(18); 5953–61. ©2011 AACR.

De Novo Biosynthetic Profiling of High Abundance Proteins in Cystic Fibrosis Lung Epithelial Cells

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210–2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [35S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFκB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that ∼30% of the 51-member hybrid high abundance CF proteome interacts with the NFκB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Protein microarray platforms for clinical proteomics

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2‐DE and/or MS need to be complemented by different kinds of technologies. The well‐known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody‐centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label‐free detection technologies, human genome‐scale numbers of analytes, and increases in analytic and bioinformatic speeds.

The significance of TNFAIP8 in prostate cancer response to radiation and docetaxel and disease recurrence

TNFAIP8 is a NF‐κB‐inducible, oncogenic molecule. Previous “promoter array” studies have identified differential methylation and regulation of TNFAIP8 in prostate epithelial and cancer cell lines. Here we demonstrate that TNFAIP8 expression is induced by androgen in hormone‐responsive LNCaP prostate cancer cells. In athymic mice bearing hormone‐refractory PC‐3 prostate tumor xenografts, intravenous treatment with a liposomal formulation of TNFAIP8 antisense oligonucleotide (LE‐AS5) caused reduced expression of TNFAIP8 in tumor tissues, and a combination of LE‐AS5 and radiation or docetaxel treatment resulted in significant inhibition of PC‐3 tumor growth as compared to single agents. The immunohistochemical evaluation of TNFAIP8 expression revealed correlation of both cytoplasmic and nuclear TNFAIP8 overexpression with high grade prostatic adenocarcinomas, while nuclear overexpression was found to be an independent predictor of disease recurrence controlling for tumor grade. Increased nuclear TNFAIP8 expression was statistically significantly associated with a 2.44 fold (95 % confidence interval: 1.01–5.91) higher risk of prostate cancer recurrence. Mechanistically, TNFAIP8 seems to function as a scaffold (or adaptor) protein. In the antibody microarray analysis of proteins associated with the TNFAIP8 immune‐complex, we have identified Karyopherin alpha2 as a novel binding partner of nuclear TNFAIP8 in PC‐3 cells. The Ingenuity Pathway Analysis of the TNFAIP8 interacting proteins suggested that TNFAIP8 influences cancer progression pathways and networks involving integrins and matrix metalloproteinases. Taken together, present studies demonstrate that TNFAIP8 is a novel therapeutic target in prostate cancer, and indicate a potential relationship of the nuclear trafficking of TNFAIP8 with adverse outcomes in a subset of prostate cancer patients.

ANXA7 expression represents hormone-relevant tumor suppression in different cancers.

Tumor suppressor function of ubiquitously expressed Annexin‐A7, ANXA7 (10q21) that is involved in exocytosis and membrane fusion was based on cancer prone phenotype in Anxa7(+/−) mice as well as ANXA7 role in human prostate and breast cancers. To clarify ANXA7 biomarker and tumor suppressor function, we analyzed its expression pattern in comparison to the prostate‐specific biomarker NKX3.1. Immunohistochemistry‐based ANXA7 and NKX3.1 protein expression was analyzed on human tissue microarrays of 4,061 specimens from a wide spectrum of the histopathologically well‐characterized tumors in different stages compared to corresponding normal tissues. Decreased ANXA7 expression was mostly associated with high invasive potential in multiple tumors. Although some metastases retained relatively high ANXA7 rates compared to primary cancer tissues, the lymph node metastases from different sites (including prostate and breast) had decreased ANXA7 expression in comparison to the intact lymphatic tissues. Major ANXA7 downregulation pattern was deviated in tumors of glandular (especially neuroendocrine) origin. ANXA7 and NKX3.1 proteins were synexpressed in the male urogenital system and adrenal gland. Gene expression profiling in prostate and breast cancers (SMD) revealed distinct hormone‐related profiles for NKX3.1 and ANXA7, where ANXA7 expression correlated with steroid sulfatase which has a pivotal role in steroidogenesis. Abundant protein presence in adrenal gland and its loss in hormone‐refractory prostate cancer indicated that ANXA7 can be relevant to steroidogenesis and androgen sensitivity in particular. With tumor suppressor pattern validated in different tumors, ANXA7 can be an attractive diagnostic and therapeutic target associated with the hormone and/or neurotransmitter‐mediated modulation of tumorigenesis.

Reverse phase protein microarray technology in traumatic brain injury.

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

Prognostic Impact of ANX7-GTPase in Metastatic and HER2-Negative Breast Cancer Patients

Purpose: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival Experimental Design: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein. Results: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study. Conclusions: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.