Momen Elshazley

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT‐5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT‐5A but not in ACC‐MESO‐1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage‐dependent and independent clonal growth in two MPM cell lines (ACC‐MESO‐1 and H290) but not in MeT‐5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC‐MESO‐1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.

TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival

TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7‐fold (P < 0.001) in non‐small cell lung cancer cell lines (n = 116) compared to normal lung controls (n = 59). In addition, small cell lung cancer cell lines (n = 29) expressed TIM at levels 2.2‐fold (P < 0.001) higher than non‐small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.

Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells

Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi‐mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM‐expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM‐expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27Kip1 upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential. (Cancer Sci 2011; 102: 1493–1500)

Febrile complications after endobronchial ultrasound-guided transbronchial needle aspiration for intra-pulmonary mass lesions of lung cancer—a series of 3 cases

Recent case reports have shown that endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for mediastinal lesions is sometimes accompanied by severe infectious complications. Here, we report 3 cases with refractory febrile complications following EBUS-TBNA for intra-pulmonary large mass lesion of lung cancer (squamous cell carcinoma, n=2; adenocarcinoma, n=1). After the EBUS-TBNA, all cases showed prolonged fever and systemic inflammation despite receiving a sufficient dose of broad-spectrum antibiotics. The presence of a low-density area inside the masses upon CT examination, suggesting necrosis, may be a predictive sign of febrile complications associated with EBUS-TBNA.

De novo childhood myelodysplastic/myeloproliferative disease with unique molecular characteristics.

Myelodysplastic/myeloproliferative uclassifiable (MDS/MPN‐U) is a rare myeloid neoplasm characterized by myelodysplasia and myeloproliferation at the time of initial presentation, which is usually a diagnosis of exclusion. The molecular pathogenesis of MDS/MPN‐U patients remains to be elucidated. Among five patients diagnosed with MDS/MPN‐U, three patients harboured RUNX1 (AML1) mutations; one carried somatic mosaicism of RUNX1 mutation with JAK2V617F mutation and one had dual RUNX1 and FLT3‐internal tandem duplication mutations with progression to acute myeloid leukaemia (AML). Germline mutation of TP53 was detected as a sole genetic lesion in one patient. JAK2V617F and somatic mosaicism of KRAS and TET2 mutations co‐existed in one patient. Otherwise, no alterations were detected in PTPN11, NRAS, CBL and ASXL1 genes. ETV6‐PDGFRB fusion transcript was not detected in all patients. Four patients recieved haematopoietic stem cell transplantation (HSCT); three patients relapsed and one achieved complete remission after three donor lymphocyte infusions. Our findings suggest that the mutational spectrum observed in childhood MDS/MPN‐U is quite different from that seen in juvenile myelomonocytic leukaemia and, to some extent, resemble chronic myelomonocytic leukaemia. Moreover, two patients had constitutional alterations of genes frequently found in AML. Further investigations are required to define the roles of these genetic alterations in the pathogenesis of childhood MDS/MPN‐U.

RUNX1 mutation associated with clonal evolution in relapsed pediatric acute myeloid leukemia with t(16;21)(p11;q22)

AbstractTLS/FUS-ERG chimeric fusion transcript resulting from translocation changes involving chromosomes 16 and 21 is a rare genetic event associated with acute myeloid leukemia (AML). The distinct t(16;21) AML subtype exhibits unique clinical and morphological features and is associated with poor prognosis and a high relapse rate; however, the underlying mechanism remains to be clarified. Recently, whole-genome sequencing revealed a large set of genetic alterations that may be relevant for the dynamic clonal evolution and relapse pathogenesis of AML. Here, we report three pediatric AML patients with t(16;21) (p11; q22). The TLS/FUS-ERG fusion transcript was detected in all diagnostic and relapsed samples, with the exception of one relapsed sample. We searched for several genetic lesions, such as RUNX1, FLT3, c-KIT, NRAS, KRAS, TP53, CBL, ASXL1, IDH1/2, and DNMT3A, in primary and relapsed AML samples. Interestingly, we found RUNX1 mutation in relapsed sample of one patient in whom cytogenetic analysis showed the emergence of a new additional clone. Otherwise, there were no genetic alterations in FLT3, c-KIT, NRAS, KRAS, TP53, CBL, ASXL1, IDH1/2, or DNMT3A. Our results suggest that precedent genetic alterations may be essential to drive the progression and relapse of t(16;21)-AML patients.

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells.

BackgroundThe role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.MethodsThe expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference–mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.ResultsMost MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.ConclusionsRNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.

Abstract 4678: Silencing of TIMELESS induces growth suppression, apoptosis and enhanced cytotoxicity of cisplatin in H157 lung cancer cells

Background: TIMELSS (TIM) is a circadian clock gene in Drosophila, but its role in mammals in circadian clock systems is not well-understood. Mammalian TIM is involved in Chk1 activation and intra-S checkpoint through genotoxic stress (Unsal-Kacmaz et.al, Molecular and Cellular Biology, 27(8), 3131-3142(2007)). TIM is also associated with doxorubicin-induced G2/M cell cycle arrest and TIM depletion sensitizes HCT116 colon cancer cells to doxorubicin-induced cytotoxicity (Yang et.al, Journal of Biological Chemistry, 285(5), 3030-3034(2010)). Mutations in TIM in breast cancer were reported (Sjoblom et.al, Science, 314(5797), 268-274(2006)). Methods and Findings: The mRNA expression levels of TIM in 17 non-small cell lung cancer (NSCLC) cell lines and 4 small cell lung cancer (SCLC) cell lines were analyzed by reverse transcriptase PCR. HCC44, H460, H157 (NSCLC), H146 and H740 (SCLC) expressed higher levels of TIM than cdk4/hTERT-immortalized normal human bronchial epithelial cell line HBEC4. Sequencing analysis of TIM revealed no mutation in 21 lung cancer cell lines. RNAi-mediated knockdown of TIM in H157 suppressed proliferation in WST-1 assay and clonogenic growth in liquid colony formation assay. Western blot analysis revealed increased level of cleaved caspase-3 after TIM-knockdown, suggesting that apoptosis was involved in growth inhibition. TIM-knockdown induced increased sensitivity to cisplatin in drug sensitivity assay using WST-1 (IC50 were 1.70μM and 0.91μM in TIM-knocked down H157 compared to 4.87μM in control). Conclusions: TIM inhibition causes growth suppression, apoptosis and enhanced cytotoxicity of cisplatin. Our results suggest that TIM inhibition has a potential utility in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4678. doi:1538-7445.AM2012-4678

Abstract C37: BMAL1 depletion represses growth of mesothelioma through induction of mitotic catastrophe

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the pleura. Asbestos exposure is considered the most important carcinogenic factor that has been mentioned in relation to MPM. Although the incidence of MPM continues to increase, it remains resistant to the currently available therapeutic modalities. Thus, identification of more efficient therapeutics based on its molecular abnormalities is highly recommended. Recently, a genome-wide expression analysis showed deregulation of the circadian rhythm pathway in MPM, including overexpression of BMAL1, a central player in the circadian clock machinery. In this study we explored the consequences of targeting BMAL1 in MPM. This was accomplished by genetic knockdown of BMAL1 with small interference RNA (siRNA) and short hairpin lentiviral expression system. The majority of MPM cell lines expressed higher levels of BMAL1 than the nontumorigenic mesothelial cell line (Met-5A). Immunohistochemical analysis revealed that BMAL1 is constitutively expressed in the cytoplasm with granular pattern in a subset of clinical MPM specimens. Long-term depletion of BMAL1 induced senescence and downregulation of IL-8 and BcL-2 in ACC-MESO-1-cells. Notably, RNAi-mediated knockdown of BMAL1 resulted in suppression of cell growth and induction of apoptosis in MPM cell lines with limited consequences in Met5A. Moreover, ACC-MESO-1 cells, which expressed the highest level of BMAL1, underwent drastic morphological changes including micronucleation, multiple nuclei, and increased cellular volume. Consistently, cell cycle profiling revealed substantial increase in apoptotic and polyploidy cells with concomitant decrease in Wee1, cyclin B, p21, and increase in cleaved caspase 3 and cyclin E proteins by Western blot analysis. Taken together, these results suggest that mitotic catastrophe could be the main cause of cells death in ACC-MESO-1 cells after BMAL1 knockdown. BMAL1 plays an important role in MPM and serves as an attractive therapeutic target for MPM with high therapeutic index. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C37.

Abstract 1657: A pivotal role for epithelial cell adhesion molecule (EpCAM) in survival of lung cancer cells

Introduction: Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers, including lung cancer, and its contribution to increased proliferation through up-regulating cell cycle accelerators such as cyclin A/E is well demonstrated in breast and gastric cancers. Nevertheless, very little is known about its role in survival of cancer cells. In addition, functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. Matrials and methods: 18 human non-small cell lung cancer cell lines and an immortalized normal human bronchial epithelial cell (HBEC4) lines were used. EpCAM expression was measured by quantitative real-time PCR (qRT-PCR) and FACS. RNA interference (RNAi)-mediated gene silencing for EpCAM was done in three non-small cell lung cancer cell and HBEC4 lines. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. Apoptosis was evaluated by annexin V/7-AAD staining. FACS with PI staining was done to examine apoptosis and cell cycle. p27 kip1 expression was evaluated by western blot analysis. In vitro invasion assay was done by using transwell chambers layered with matrigel and invading cells were stained and counted. Results: We analyzed the expression of EpCAM in a panel of lung cancer cell and HBEC4 lines and found that most of lung cancer cell lines expressed EpCAM. EpCAM knockdown suppressed proliferation and clonogenic growth of two EpCAM-expressing lung cancer cell lines in anchorage dependent and independent conditions. EpCAM knockdown suppressed invasiveness in a highly invasive line but not in a lowly invasive cell line. In addition, EpCAM knockdown induced massive apoptosis in both cell lines as well as another EpCAM-expressing lung cancer cell line but to a much lesser extent in HBEC4 line. EpCAM knockdown caused lung cancer cells to have increased responsiveness to contact inhibition in part through up-regulating p27 kip1 cyclin dependent kinase inhibitor. Conclusion: These results demonstrate that EpCAM substantially contributes to the pathogenesis of lung cancer, especially in its survival, and the development of EpCAM-targeted therapy for lung cancer may have promise. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1657. doi:10.1158/1538-7445.AM2011-1657