Ala-Eddin Al Moustafa

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin β1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin β1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells.

Identification of genes associated with head and neck carcinogenesis by cDNA microarray comparison between matched primary normal epithelial and squamous carcinoma cells.

In order to identify genes involved in head and neck carcinogenesis, we compared the gene expression profile in matched primary normal epithelial cells and primary head and neck cancer cells from the same patients. A cDNA microarray analysis consisting of 12 530 human genes revealed significant changes in the expression of 213 genes, with 91 genes being up-regulated and 122 being down-regulated. This comprehensive list of genes includes those associated with signal transduction (growth factors), cell structure, cell cycle, transcription, apoptosis, and cell–cell adhesion. Further analysis of nine genes involved in cell–cell interaction, using Western blot and/or reverse transcription (RT)–PCR of four paired cell lines supported the reliability of our microarray analysis. More specifically, our study provides the first evidence that claudin-7 and connexin 31.1 are down-regulated in head and neck squamous cell carcinomas (HNSCC) compared to normal cells. These findings provide a large body of information regarding gene expression profiles associated with head and neck carcinogenesis, and also represent a source of potential targets for HNSCC prevention and/or therapeutics.

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.

E6/E7 of HPV Type 16 Promotes Cell Invasion and Metastasis of Human Breast Cancer Cells

Human papillomaviruses (HPVs) could be important risk factors for breast carcinogenesis and metastasis, as roughly 50% of breast cancers are positive for high-risk HPVs. To determine the role of high-risk HPVs in human breast carcinogenesis and metastasis, we examined the effect of E6/E7 of HPV type 16 in two non-invasive breast cancer cell lines, MCF7 and BT20. We report that E6/E7 of HPV type 16 induces cell invasive and metastatic abilities of MCF7 and BT20 in vitro and in vivo, respectively, in comparison with the wild type cells. This is accompanied by an up-regulation of Id-1, a family member of helix-loop-helix (HLH) transcription factors, in MCF7 and BT20 cell lines which express E6/E7. Earlier studies have reported that Id-1 regulates cell invasion and metastasis of human breast cancer cells. To gauge the role of Id-1 in cell invasion and metastasis induced by E6/E7 of HPV type 16, we investigated the effect of E6/E7 in mouse normal embryonic fibroblast (NEF) and knockout Id-1 (Id-1-/-) cells. We establish that E6/E7 induces cell invasive ability in NEF but not Id-1-/- cells; moreover, we were able to inhibit the invasion ability of MCF7-E6/E7 and BT20-E6/E7 using Id-1 antisense retroviruses. Furthermore, we report that E6/E7 oncoproteins up-regulate Id-1 promoter activity in MCF7 and BT20 cells. We also found that HPV type 16 is present in all invasive and metastatic breast cancer and less frequently in in-situ breast cancer as opposed to normal mammary tissue. In parallel, we demonstrate that Id-1 over-expression is correlated with the presence of HPV type 16 in human invasive and metastatic breast cancer. These data suggest that high-risk HPV infections can induce cell invasion and metastasis in breast cancer through Id-1 regulation.

Cyclin D1 is essential for neoplastic transformation induced by both E6/E7 and E6/E7/ErbB-2 cooperation in normal cells.

More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial (NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic fibroblast (NEF), wild type (wt), and knockout cyclin D1 (D1−/−) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of cyclin D1−/− cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.

Identification of deregulated genes by single wall carbon-nanotubes in human normal bronchial epithelial cells

To identify genes affected by single-walled carbon nanotubes (SWCNTs) in human normal lung cells, we compared the gene expression profiles of untreated human normal bronchial epithelial (HNBE) cells to profiles of HNBE cells treated with SWCNTs. A complementary DNA microarray analysis consisting of 54,675 human genes revealed marked changes in the expression of 14,294 genes, with 7,029 genes being upregulated and 7,265 being downregulated. This comprehensive list of genes included those associated with cell cycle, apoptosis, cell survival, cell adhesion and motility, signal transduction, and transcription regulation. Additional analysis of 19 genes using reverse transcription-polymerase chain reaction confirmed the microarray analysis. More specifically, our study demonstrates to our knowledge for the first time, evidence that 9 of the 19 genes (most of which encode cell apoptotic, signal transduction, and transcription regulator products) are upregulated in the SWCNTs-treated HNBE cells as compared with untreated cells, whereas the remaining 10 of the 19 (involved in cell adhesion and motility, cell proliferation, and cell survival) are downregulated in SWCNTs-treated HNBE cells in comparison with untreated controls. These findings provide a large body of information regarding gene expression profiles associated with SWCNTs exposure in human lung bronchial epithelial cells, and also represent a source to investigate the mechanism of the effect of SWCNTs in human normal lung cells. From the clinical editor: In this study, the gene expression profile of human normal bronchial epithelial cells was compared with single-wall carbon nanotubes-treated cells. A cDNA microarray analysis consisting of 54,675 human genes revealed significant changes in the expression of 14,294 genes, with 7,029 genes being up-regulated and 7,265 being down-regulated. This serves as a first step in clarification of mechanisms of action and to investigate toxicity in this model.

ErbB receptors and epithelial-cadherin-catenin complex in human carcinomas.

The ErbB family of receptor tyrosine kinases have important roles in maintaining normal epithelial cell function. The ErbBs are involved in the interaction between cells and cell-matrix adhesion molecules and have proven critical in maintaining the integrity of the epithelial cell environment. Deregulation of these tyrosine receptors has been associated with several human diseases. In particular, the expression or activation of epidermal growth factor receptor (EGFR) and ErbB2 is altered in many epithelial tumors. Epithelial (E)-cadherin is another major molecule expressed by epithelial cells. To create efficient cell-cell adhesion, E-cadherin couples its cytoplasmic domain to catenins and the actin cytoskeleton. The loss of intercellular adhesion appears to be a fundamental aspect of the neoplastic phenomena. In addition, EGFR and ErbB2 signaling associated with the E-cadherin-catenin complex has been demonstrated in normal and cancer cells. This signaling is involved in regulating cell adhesion and the invasive growth of cancers. This article provides an overview of the interaction between the ErbB tyrosine receptors and the E-cadherin-catenin complex in human carcinomas.

Water pipe smoking and human oral cancers.

While cigarette smoking is recognized as an important risk factor in human oral cancers, the effect of water pipe smoking (WPS) on these cancers is not known. WPS is very common in the young adult population, especially in the Middle East, and has been associated with several respiratory problems. However, to date, there have been no studies examining the association between WPS and the progression of human oral cancers. Currently, the role of WPS in human oral cancers remains uncertain because of the limited number of investigations. This raises the question of whether WPS plays a significant role in the development of human oral carcinomas. In this paper, we propose the hypothesis that human oral normal epithelial cells are vulnerable to persistent WPS; moreover, WPS could play an important role in the initiation of a neoplastic transformation of human normal oral epithelial cells. Therefore, we believe that an international collaboration of epidemiological and clinical studies as well as cellular and molecular biology investigations is necessary to answer this important question.

Association between human papillomavirus and Epstein-Barr virus infections in human oral carcinogenesis

Infection by high-risk human papillomaviruses (HPVs) and Epstein-Barr virus (EBV) are very frequent in the adult human population, and have been associated with several human carcinomas, especially oral cancers. However, a small number of studies have examined the association between high-risk HPV and EBV in the progression of human oral cancers. Currently, the role of high-risk HPV and EBV co-infections in human oral cancers, particularly nasopharyngeal carcinomas, remain uncertain because of the limited number of investigations. This raises the question whether high-risk HPV and EBV co-infections play a significant role in the development of human nasopharyngeal carcinomas. In this paper, we propose the hypothesis that human oral normal epithelial cells, especially nasopharyngeal cells, are very susceptible to persistent HPV and EBV co-infections; therefore, high-risk HPV and EBV co-infections play an important role in the initiation of a neoplastic transformation of human oral epithelial cells. We believe that significant studies, using different cells and animal models as well as clinical samples, are necessary to answer these important questions.

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

Overexpression of the erbB-2 tyrosine kinase receptor, p185erbB-2, is a common alteration in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis and a tumor drug resistance phenotype. In this study, we have examined the consequences of erbB-2 depletion on DNA repair, cell cycle, and apoptosis using a panel of NSCLC cell lines constitutively overexpressing erbB-2 receptor. Depletion of the erbB-2 was achieved using the tyrosine kinase inhibitor CP127,374 which promotes erbB-2 degradation. Treatment with CP127,374 concentrations which deplete erbB-2 and inhibit tyrosine phosphorylation resulted in downregulation of DNA repair mechanisms and cell accumulation at G1 phase of the cell cycle. G1 arrest was observed in cells with mutated p53 as well as cells lacking p53 protein, suggesting a p53-independent mechanisms. NSCLC cells which overexpress erbB-2 were more resistant to cisplatin-induced cytotoxicity in comparison to cells expressing low levels of erbB-2. Treatment with CP127,374 alone did not result in any induction of apoptosis. A combination of CP127,374 and cisplatin, however, was more potent in cell growth inhibition and induction of apoptosis compared to treatment with cisplatin alone. Together, our results further support a pivotal role of erbB-2 signaling in the regulatory balance between DNA repair, cell cycle checkpoints and apoptosis; all these mechanisms are essential determinants for tumor cell destiny following chemotherapy stress.