Naciba Benlimame

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3–induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB–FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

E6/E7 proteins of HPV type 16 and ErbB-2 cooperate to induce neoplastic transformation of primary normal oral epithelial cells

Head and neck squamous cell carcinomas (HNSCC) are characterized by a marked propensity for local invasion and spread to cervical lymph nodes, with distant metastases developing in 30–40% of cases. HPV-16 is an important risk factor for HNSCC. How HPV enhances susceptibility to HNSCC is not fully understood, but seems to involve cofactors. In this study, we examined the effect of the cooperation between HPV-16 and the tyrosine kinase receptor ErbB-2 on E-cadherin/catenin complex patterns and neoplastic transformation of human normal oral epithelial (NOE) cells. We report that overexpression of ErbB-2 or E6/E7 alone does not affect E-cadherin/catenin complex patterns nor does it induce cell transformation of NOE cells. In contrast, coexpression of E6/E7 and ErbB-2 downregulates E-cadherin and catenin expression. This is accompanied by cytoplasmic localization of E-cadherin, as well as nuclear translocation of α, β, and γ-catenins. Furthermore, we demonstrate that E6/E7 cooperate with overexpressed ErbB-2 to induce tumor formation in nude mice and to upregulate cyclin D1 and c-myc expression. Our data suggest that E6/E7 cooperate with ErbB-2 in head and neck carcinogenesis, at least in part, via the conversion of β-catenin from a cell adhesion to a nuclear function, that is, to act as a potential transcriptional regulator. This conversion leads to the upregulation of cyclin D1, c-myc and other oncoproteins necessary for alteration of the E-cadherin/catenin complex and cell transformation of NOE cells.

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor’s biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Methods for Sample Acquisition and Processing of Serial Blood and Tumor Biopsies for Multicenter Diffuse Large B-cell Lymphoma Clinical Trials

Increasingly, targeted therapies are being developed to treat malignancies. To define targets, determine mechanisms of response and resistance, and develop biomarkers for the successful investigation of novel therapeutics, high-quality tumor biospecimens are critical. We have developed standard operating procedures (SOPs) to acquire and process serial blood and tumor biopsies from patients with diffuse large B-cell lymphoma enrolled in multicenter clinical trials. These SOPs allow for collection and processing of materials suitable for multiple downstream applications, including immunohistochemistry, cDNA microarrays, exome sequencing, and metabolomics. By standardizing these methods, we control preanalytic variables that ensure high reproducibility of results and facilitate the integration of datasets from such trials. This will facilitate translational research, better treatment selection, and more rapid and efficient development of new drugs. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2688–93. ©2014 AACR.

A novel parasite-derived suicide gene for cancer gene therapy with specificity for lung cancer cells

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.

Abstract 64: Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials.

Introduction: Personalized medicine strategies targeting specific aberrations unique to an individual tumor, are promising to become a new paradigm in the fight against cancer. Such tailored approaches rely on the thorough profiling of malignant tissues by high-throughput technologies followed by selection of appropriate treatment targeting tumor-specific aberrations. One fundamental challenge, whose resolution is necessary for any successful personalized medicine endeavor, is isolation of useful amounts of good quality molecular material for reliable tumor characterization in downstream assays. Here, we describe our standardized protocol for biopsy collection and results from a multi-center phase II clinical trial investigating the use of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma (NCT01238692). Results: Biopsies are performed with patient consent, before treatment initiation and after 15 days of treatment, in patients with safely accessible lesions. Four needle core biopsies are collected at each time point using standard operating procedures to limit pre-analytical variability. Of these, one is preserved in formalin for immunohistochemical (IHC) analyses, while the remaining three are pooled together in RPMI 1640 media. These samples are sent to a central laboratory for further processing. The needle cores in RPMI 1640 undergo B-cell purification using a negative selection kit (StemCell Technologies). Isolation of tumor RNA and DNA is performed using a commercially available kit (Qiagen). Of the 23 patients currently enrolled, 16 (70%) have had a pre-treatment biopsy performed and of these, 7 (44% of those biopsied, 30% of all patients) have also had a post-treatment biopsy, demonstrating the feasibility of tissue collection at participating sites. We assess the effect of transport on the quality and yield of RNA and DNA as well as differences in quality and yield between pre-treatment biopsy vs. biopsies at day 15. The material isolated from these biopsies is evaluated for suitability in downstream applications, namely IHC, gene expression microarray and DNA exome sequencing. Conclusions: The development of standard operating procedures for the collection and processing of biospecimens is essential to control for pre-analytical variability inherent to multicenter trials. Our experience shows that this is both feasible and crucial to understanding of tumor molecular profile changes associated with treatment. Our protocol allows extraction of RNA and DNA of sufficient quality and quantity to permit downstream multi-dimensional analysis. Patient acceptance of research biopsies has been high although the rate of pre-treatment biopsies is greater than that of post-treatment biopsies. Finally, our work demonstrates that the timing of post-treatment biopsies is critical. Citation Format: Torsten H. Nielsen, Zuanel Diaz, Rosa Christodoulopoulos, Lu Yao, Samia Qureshi, Naciba Benlimame, Errol Camlioglu, Michael Crump, Ryan D. Morin, Nathalie Johnson, Tina P. Haliotis, Wilson H. Miller, Sarit Assouline, Koren K. Mann. Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2013-64