Pinaki Bose

REAP: A two minute cell fractionation method

BackgroundThe translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT.FindingsWe have developed a R apid, E fficient A nd P ractical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.ConclusionsThis method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.

Tethering by lamin A stabilizes and targets the ING1 tumour suppressor

ING proteins interact with core histones through their plant homeodomains (PHDs) and with histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes to alter chromatin structure. Here we identify a lamin interaction domain (LID) found only in ING proteins, through which they bind to and colocalize with lamin A. Lamin knockout (LMNA−/−) cells show reduced levels of ING1 that mislocalize. Ectopic lamin A expression increases ING1 levels and re-targets it to the nucleus to act as an epigenetic regulator. ING1 lacking the LID does not interact with lamin A or affect apoptosis. In LMNA−/− cells, apoptosis is not affected by ING1. Mutation of lamin A results in several laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), a severe premature ageing disorder. HGPS cells have reduced ING1 levels that mislocalize. Expression of LID peptides to block lamin A–ING1 interaction induces phenotypes reminiscent of laminopathies including HGPS. These data show that targeting of ING1 to the nucleus by lamin A maintains ING1 levels and biological function. Known roles for ING proteins in regulating apoptosis and chromatin structure indicate that loss of lamin A–ING interaction may be an effector of lamin A loss, contributing to the HGPS phenotype.

Head and neck cancer: from anatomy to biology.

The 20th century saw great advances in anatomy‐based (surgery and radiotherapy) and chemotherapy approaches for treating head and neck squamous cell carcinoma (HNSCC) and improving quality of life (QoL). However, despite these advances, the survival rate in HNSCC remains at ∼50%. Front‐line treatments often cause severe toxicity and debilitating long‐term impacts on QoL. In recent decades, dramatic advances have been made in our knowledge of fundamental tumor biology and signaling pathways that contribute to oncogenesis and cancer progression. These insights are presenting unprecedented opportunities to develop more effective and less toxic treatments that are specific to particular molecular targets. This review discusses some of the major, potentially targetable, molecular pathways associated with head and neck carcinogenesis. We present the general mechanism underlying the functional components for each signaling pathway, discuss how these components are aberrantly regulated in HNSCC and describe their potential as therapeutic targets. We have restricted our discussion to “drug‐able targets” such as oncogenes including those associated with HPV, tumor hypoxia and microRNAs and present these changes in the context of HNSCC patient care. The specific targeting of these pathways to achieve cancer control/remission and reduce toxicity is now challenging conventional treatment paradigms in HNSCC. This new “biologic era” is transforming our ability to target causal pathways and improve survival outcomes in HNSCC.

High stromal carbonic anhydrase IX expression is associated with nodal metastasis and decreased survival in patients with surgically-treated oral cavity squamous cell carcinoma

Every year, approximately 25,000 patients are diagnosed with oral cavity squamous cell carcinoma (OCSCC) in the USA. The 5-year survival rate for OCSCC is approximately 40%. Intratumoral hypoxia confers poor prognosis and treatment failure but direct tumor oxygen measurement is challenging. Carbonic anhydrase IX (CAIX) is a marker of tissue hypoxia and we have recently shown that stromal CAIX is associated with reduced survival in patients with HPV-negative head and neck cancer. We examined the importance of this observation in OCSCC patients. We identified patients diagnosed and treated with OCSCC in Calgary (Alberta, Canada) between 1998 and 2005. Clinical and pathologic data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of archived tumor tissue. Stromal CAIX expression was assessed by quantitative immunohistochemistry (AQUA-HistoRx). The primary endpoint was disease-specific survival. We identified 102 patients with OCSCC; 87 patients had surgery as their primary treatment and adequate tumor tissue for TMA construction was available for all patients. CAIX expression was evaluable for 61 patients. High (top quartile) stromal CAIX expression was associated with significantly reduced 5-year disease-specific survival compared to low stromal CAIX expression (p<0.006). This study confirms our previously reported association between high stromal CAIX expression and significantly reduced overall survival in an independent, predominantly p16-negative, cohort of surgically treated OCSCC. Assessment of stromal CAIX expression could identify patients with the least favorable prognosis and inform therapeutic strategies in OCSCC.

Basal Ki67 expression measured by digital image analysis is optimal for prognostication in oral squamous cell carcinoma

AIM The prognostic significance of Ki67 expression in cancers, including oral squamous cell carcinoma (OSCC), is unclear. This may be partly attributed to the lack of consensus surrounding the optimal approach for measuring tumour Ki67 expression. The aim of this study was to evaluate the association between different measures of Ki67 expression and disease-specific survival (DSS) in OSCC. METHODS Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded (FFPE) pre-treatment tumour tissue obtained from 121 OSCC patients diagnosed between 1998 and 2006. Ki67 expression was quantified using fluorescence immunohistochemistry (IHC) and AQUAnalysis® in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Intensity and percentage-based approaches for Ki67 scoring were tested for their association with survival. RESULTS Ki67 scores obtained from intensity and percentage-based approaches had similar associations with prognosis. We also found that high basal (lowest observed in triplicate cores) Ki67 expression was more strongly associated with improved 5-year disease-specific survival than hot-spot and average Ki67 measurements. The association of high basal Ki67 expression with improved prognosis was most pronounced in patients who received postoperative radiation. Cox proportional hazards analysis showed that the basal Ki67 expression is an independent prognostic marker in our OSCC cohort when adjusted for pathological T-stage, nodal status and treatment. CONCLUSIONS Our study provides a framework for reaching a consensus on the optimal approach for measuring Ki67 expression in cancers. Our results suggest that rigorous comparisons of measurement approaches should be applied in a tumour-type and treatment-specific manner to enhance the clinical application of Ki67 assessment.

Precursor States of Brain Tumor Initiating Cell Lines Are Predictive of Survival in Xenografts and Associated with Glioblastoma Subtypes

Summary In glioblastoma multiforme (GBM), brain-tumor-initiating cells (BTICs) with cancer stem cell characteristics have been identified and proposed as primordial cells responsible for disease initiation, recurrence, and therapeutic resistance. However, the extent to which individual, patient-derived BTIC lines reflect the heterogeneity of GBM remains poorly understood. Here we applied a stem cell biology approach and compared self-renewal, marker expression, label retention, and asymmetric cell division in 20 BTIC lines. Through cluster analysis, we identified two subgroups of BTIC lines with distinct precursor states, stem- or progenitor-like, predictive of survival after xenograft. Moreover, stem and progenitor transcriptomic signatures were identified, which showed a strong association with the proneural and mesenchymal subtypes, respectively, in the TCGA cohort. This study proposes a different framework for the study and use of BTIC lines and provides precursor biology insights into GBM.

MRI Texture Analysis Predicts p53 Status in Head and Neck Squamous Cell Carcinoma

BACKGROUND AND PURPOSE: Head and neck cancer is common, and understanding the prognosis is an important part of patient management. In addition to the Tumor, Node, Metastasis staging system, tumor biomarkers are becoming more useful in understanding prognosis and directing treatment. We assessed whether MR imaging texture analysis would correctly classify oropharyngeal squamous cell carcinoma according to p53 status. MATERIALS AND METHODS: A cohort of 16 patients with oropharyngeal squamous cell carcinoma was prospectively evaluated by using standard clinical, histopathologic, and imaging techniques. Tumors were stained for p53 and scored by an anatomic pathologist. Regions of interest on MR imaging were selected by a neuroradiologist and then analyzed by using our 2D fast time-frequency transform tool. The quantified textures were assessed by using the subset-size forward-selection algorithm in the Waikato Environment for Knowledge Analysis. Features found to be significant were used to create a statistical model to predict p53 status. The model was tested by using a Bayesian network classifier with 10-fold stratified cross-validation. RESULTS: Feature selection identified 7 significant texture variables that were used in a predictive model. The resulting model predicted p53 status with 81.3% accuracy (P < .05). Cross-validation showed a moderate level of agreement (κ = 0.625). CONCLUSIONS: This study shows that MR imaging texture analysis correctly predicts p53 status in oropharyngeal squamous cell carcinoma with ∼80% accuracy. As our knowledge of and dependence on tumor biomarkers expand, MR imaging texture analysis warrants further study in oropharyngeal squamous cell carcinoma and other head and neck tumors.

Bax expression measured by AQUAnalysis is an independent prognostic marker in oral squamous cell carcinoma

BackgroundResistance to apoptosis is a hallmark of cancer and proteins regulating apoptosis have been proposed as prognostic markers in several malignancies. However, the prognostic impact of apoptotic markers has not been consistently demonstrated in oral squamous cell carcinoma (OSCC). This inconsistency in reported associations between apoptotic proteins and prognosis can be partly attributed to the intrinsic low resolution and misclassification associated with manual, semi-quantitative methods of biomarker expression measurement. The aim of this study was to examine the association between apoptosis-regulating proteins and clinical outcomes in oral squamous cell carcinoma (OSCC) using the quantitative fluorescence immunohistochemistry (IHC) based AQUAnalysis technique.MethodsSixty-nine OSCC patients diagnosed between 1998–2005 in Calgary, Alberta, Canada were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded pre-treatment tumour tissue. Bax, Bcl-2 and Bcl-XL protein expression was quantified using fluorescent IHC and AQUA technology in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Survival was analyzed using Kaplan-Meier plots and the Cox proportional hazard model.ResultsBax expression was predominantly nuclear in OCSE and almost exclusively cytoplasmic in OSCC. No similar differences in localization were observed for Bcl-2 or Bcl-XL. Only Bax expression associated with disease-specific survival (DSS), with 5-year survival estimates of 85.7% for high Bax versus 50.3% for low Bax (p = 0.006), in univariate analysis. High Bax expression was also significantly associated with elevated Ki67 expression, indicating that increased proliferation might lead to an improved response to radiotherapy in patients with elevated Bax expression. In multivariate analyses, Bax protein expression remained an independent predictor of DSS in OSCC [HR 0.241 (0.078-0.745), p = 0.013].ConclusionsThe AQUA technique used in our study eliminates observer bias and provides reliable and reproducible estimates for biomarker expression. AQUA also provides essential measures of quality control that cannot be achieved with manual biomarker scoring techniques. Our results support the use of Bax protein expression as a prognostic marker in conjunction with other clinico-pathological variables when designing personalized treatment strategies for OSCC patients.

ING1 induces apoptosis through direct effects at the mitochondria

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.

Specific and Sensitive Hydrolysis Probe-Based Real-Time PCR Detection of Epidermal Growth Factor Receptor Variant III in Oral Squamous Cell Carcinoma

Background The tumor-specific EGFR deletion mutant, EGFRvIII, is characterised by ligand-independent constitutive signalling. Tumors expressing EGFRvIII are resistant to current EGFR-targeted therapy. The frequency of EGFRvIII in head and neck squamous cell carcinoma (HNSCC) is disputed and may vary by specific sub-site. The purpose of this study was to measure the occurrence of EGFRvIII mutations in a specific HNSCC subsite, oral squamous cell carcinoma (OSCC), using a novel real-time PCR assay. Methodology Pre-treatment Formalin Fixed Paraffin Embedded (FFPE) cancer specimens from 50 OSCC patients were evaluated for the presence of EGFRvIII using a novel hydrolysis probe-based real-time PCR assay. EGFR protein expression in tumor samples was quantified using fluorescent immunohistochemistry (IHC) and AQUA® technology. Principal findings We detected EGFRvIII in a single OSCC patient in our cohort (2%). We confirmed the validity of our detection technique in an independent cohort of glioblastoma patients. We also compared the sensitivity and specificity of our novel real-time EGFRvIII detection assay to conventional RT-PCR and direct sequencing. Our assay can specifically detect EGFRvIII and can discriminate against wild-type EGFR in FFPE tumor samples. AQUAnalysis® revealed that the presence of EGFRvIII transcript is associated with very high EGFR protein expression (98th percentile). Contrary to previous reports, only 44% of OSCC over-expressed EGFR in our study. Conclusion and Significance Our results suggest that the EGFRvIII mutation is rare in OSCC and corroborate previous reports of EGFRvIII expression only in tumors with extreme over-expression of EGFR. We conclude that EGFRvIII-specific therapies may not be ideally suited as first-line treatment in OSCC. Furthermore, highly specific and sensitive methods, such as the real-time RT-PCR assay and AQUAnalysis® described here, will provide accurate assessment of EGFR mutation frequency and EGFR expression, and will facilitate the selection of optimal tailored therapies for OSCC patients.