Siham Sabri

Spontaneous Epithelial-Mesenchymal Transition and Resistance to HER-2-Targeted Therapies in HER-2-Positive Luminal Breast Cancer

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.

O6-Methylguanine-DNA Methyltransferase is a Novel Negative Effector of Invasion in Glioblastoma Multiforme

The dismal prognosis of glioblastoma multiforme (GBM) is mostly due to the high propensity of GBM tumor cells to invade. We reported an inverse relationship between GBM angiogenicity and expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. In the present study, given the major role of angiogenesis and invasion in GBM aggressiveness, we aimed to investigate the relationship between MGMT expression and GBM invasion. Stable overexpression of MGMT in the U87MG cell line significantly decreased invasion, altered expression of invasion-related genes, decreased expression of α5β1 integrin and focal adhesion kinase, and reduced their spindle-shaped morphology and migration compared with the empty vector control. Conversely, short hairpin RNA-mediated stable knockdown of MGMT or its pharmacologic depletion in the MGMT-positive T98G cell line were required for increased invasion. The inverse relationship between MGMT and invasion was further validated in primary GBM patient-derived cell lines. Using paraffin-embedded tumors from patients with newly diagnosed GBM (n = 59), tumor MGMT promoter hypermethylation (MGMT gene silencing) was significantly associated with increased immunohistochemical expression of the proinvasive matricellular protein secreted protein acidic and rich in cysteine (SPARC; P = 0.039, χ2 test). Taken together, our findings highlight for the first time the role of MGMT as a negative effector of GBM invasion. Future studies are warranted to elucidate the role of SPARC in the molecular mechanisms underlying the inverse relationship between MGMT and GBM invasion and the potential use of MGMT and SPARC as biomarkers of GBM invasion. Mol Cancer Ther; 11(11); 2440–50. ©2012 AACR.

Differential response to ablative ionizing radiation in genetically distinct non-small cell lung cancer cells

ABSTRACT Stereotactic ablative radiotherapy (SABR) has emerged as a highly promising treatment for medically inoperable early-stage non-small cell lung cancer patients. Treatment outcomes after SABR have been excellent compared to conventional fractionated radiotherapy (CFRT). However, the biological determinants of the response to ablative doses of radiation remain poorly characterized. Furthermore, theres little data on the cellular and molecular response of genetically distinct NSCLC subtypes to radiation. We assessed the response of 3 genetically distinct lung adenocarcinoma cell lines to ablative and fractionated ionizing radiation (AIR and FIR). We studied clonogenic survival, cell proliferation, migration, invasion, apoptosis and senescence. We also investigated the effect of AIR and FIR on the expression of pro-invasive proteins, epithelial-to-mesenchymal transition (EMT), extracellular signal-regulated kinases (ERK1/2) and the transmembrane receptor cMET. Our findings reveal that AIR significantly reduced cell proliferation and clonogenic survival compared to FIR in A549 cells only. This differential response was not observed in HCC827 or H1975 cells. AIR significantly enhanced the invasiveness of A549 cells, but not HCC827 or H1975 cells compared to FIR. Molecular analysis of pathways involved in cell proliferation and invasion revealed that AIR significantly reduced phosphorylation of ERK1/2 and upregulated cMET expression in A549 cells. Our results show a differential proliferative and invasive response to AIR that is dependent on genetic subtype and independent of intrinsic radioresistance. Further examination of these findings in a larger panel of NSCLC cell lines and in pre-clinical models is warranted for identification of biomarkers of tumor response to AIR.

CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide

Background Cut Like homeobox 1 (CUX1), which encodes an auxiliary factor in base excision repair, resides on 7q22.1, the most frequently and highly amplified chromosomal region in glioblastomas. The resistance of glioblastoma cells to the mono-alkylating agent temozolomide is determined to some extent by the activity of apurinic/apyrimidinic endonuclease 1 (APE1). Methods To monitor the effect of CUX1 and its CUT domains on APE1 activity, DNA repair assays were performed with purified proteins and cell extracts. CUX1 protein expression was analyzed by immunohistochemistry using a tumor microarray of 150 glioblastoma samples. The effect of CUX1 knockdown and overexpression on the resistance of glioblastoma cell lines to temozolomide was investigated. Results We show that CUT domains stimulate APE1 activity. In agreement with these findings, CUX1 knockdown causes an increase in the number of abasic sites in genomic DNA and a decrease in APE1 activity as measured in cell extracts. Conversely, ectopic CUX1 expression increases APE1 activity and lowers the number of abasic sites. Having established that CUX1 is expressed at high levels in most glioblastomas, we next show that the resistance of glioblastoma cells to temozolomide and to a combined treatment of temozolomide and ionizing radiation is reduced following CUX1 knockdown, but increased by overexpression of CUX1 or a short protein containing only 2 CUT domains, which is active in DNA repair but devoid of transcriptional activity. Conclusion These findings indicate that CUX1 expression level impacts on the response of glioblastoma cells to treatment and identifies the CUT domains as potential therapeutic targets.

Abstract 4235: Anti-proliferative effects of ZR2002, a novel combi-molecule with EGFR/DNA binary targeting properties compared to Gefitinib in glioblastoma cell lines and brain tumor stem cells

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor in adults with a survival of only 12-15 months. Brain tumor stem cells (BTSCs) contribute to tumor initiation, progression, chemo- and radioresistance, which accounts for tumor recurrence and treatment failure. Amplification of epidermal growth factor receptor (EGFR) is one of the most common genetic alterations associated with GBM aggressiveness. EGFR variant III (EGFRvIII) is an activating mutation that accounts for 60% of EGFR mutations in patients whose tumors show amplification of wild type EGFR. Despite the evidence that EGFR-induced pathway represents an attractive therapeutic target in GBM, gefitinib (Iressa™, ZD1839), an orally active, selective EGFR-tyrosine kinase inhibitor showed only a limited potency in clinical trials. ZR2002, a prototype of ‘combi-molecule’ capable of generating the binary EGFR/DNA targeting activity without requirement for hydrolytic cleavage has not been tested against BTSC. Given the ability of ZR2002 to concomitantly induce DNA damage and block EGFR-mediated signaling, we hypothesized that it would exert greater anti-proliferative activity than gefitinib in GBM tumor cell lines and BTSCs. To investigate our hypothesis, we used isogenic cell lines: U87EGFR wild-type (wt) and U87EGFRvIII (over-expressing EGFRvIII), U87MG (parental cell line) in addition to T98G, a GBM cell line known to express high levels of EGFR and BTSCs derived from patients newly diagnosed with GBM. BTSCs were cultured as neurospheres in stem cell media supplemented with growth factors (EGF, FGF), heparin and proliferation supplement. We examined the half maximal inhibitory concentration (IC50) of ZR2002 compared to gefitinib using MTT proliferation assay. Our results show that all cell lines tested were resistant to gefitinib when compared to ZR2002, which showed sub-micromolar potency in all tested cell lines following a short 2 h drug exposure. IC50s for T98G, U87-MG and U87-EGFRIII treated with ZR2002 were 0.019 μM, 0.048 μM and 0.073 μM, respectively. We further investigated the effect of ZR2002 in four different BTSC cell lines 48EF, OPK111, OPK164 and OPK161 and showed that the IC50s were in a similar range 0.027 μM, 0.014 μM, 0.089 μM and 0.019 μM, respectively. While GBM cell lines and BTSCs are highly resistant to gefitinib, they showed a greater sensitivity to ZR2002 (IC50 is less than 0.1 μM). These findings pave the way for developing single molecules with dual therapeutic modalities as a new strategy for GBM treatment. Additional pre-clinical studies are ongoing to evaluate the ability of ZR2002 to cross the blood brain barrier and its efficacy in an orthotopic tumor brain model. This research is funded by the Canadian Cancer Society grant #70217. Citation Format: Zeinab Sharifi, Jean-Claude Bertrand, Kevin Petrecca, Elliot Goodfellow, Bassam Abdulkarim, Siham Sabri. Anti-proliferative effects of ZR2002, a novel combi-molecule with EGFR/DNA binary targeting properties compared to Gefitinib in glioblastoma cell lines and brain tumor stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2015-4235

Abstract 865: Evolving biological and clinical concepts of radiation delivery in NSCLC: response to ablative versus fractionated radiotherapy

Non-small cell lung cancer (NSCLC) patients account for over 80% of all lung cancer cases and have a poor outcome with current radiotherapy regimens. NSCLC has been the subject of many studies characterizing mechanisms of initiation, proliferation, invasion and treatment response. Recent trends in radiotherapy show an increase in the use of ablative radiotherapy (ART) in inoperable small size tumors. Clinical studies have shown response rates up to 90% in NSCLC patients treated with ART compared to 15-20% in patients treated with conventional fractionated radiotherapy (FRT). However, the biological determinants of this response have not been investigated and recent analysis of patterns of failure in patients treated with ART show an increased tendency towards distant metastatic recurrence. We investigated the biological response of NSCLC cell lines with different molecular subtypes including, A549, H1975 and HCC827 to ART and FRT. Radiation doses of 8Gy and 12Gy were delivered in multiple fractions or single fraction. The response to radiation was investigated using several cellular assays including clonogenic survival, cell proliferation, matrigel invasion, wound-healing, morphological characterization and senescence-associated beta-galactosidase. ART significantly reduced cell proliferation and clonogenic survival compared to FRT in A549 cells. In addition, a significant increase in the number of senescent cells as well as large, polynucleated cells was observed in the ART-treated group compared to the FRT-treated group. This differential response to delivery approach (ART vs FRT) was not observed in HCC827 or H1975 cells which harbor EGFR mutations. Both ART and FRT inhibited cell proliferation and clonogenic survival to similar levels in HCC827 and H1975 cells. In contrast to reduced cell proliferation and clonogenicity, ART significantly increased the invasive phenotype of A549 cells, but not HCC827 or H1975 cells compared to FRT. Western blot analysis of several markers of invasion, including cMET, AKT, ERK, SPARC and FAK revealed a significant down regulation of SPARC in A549 cells exposed to ART, but not FRT. Further analysis of cells in Boyden chambers showed that ART-induced senescent cells are capable of migration/invasion. Our results unequivocally demonstrate that response to ART is cell-line dependent. A549 cells, which harbor wild-type EGFR have a differential response to radiotherapy based on delivery approach. Furthermore, A549 cell exposed to ART have significantly increased invasive and migratory capacity compared to FRT. Our findings suggest that the extracellular matrix glycoprotein, SPARC is involved in the modulation of radiation-induced invasion/migration. These findings can have significant implications for NSCLC patients undergoing ART and underscore the importance of understanding the underlying biology for effective disease management. Citation Format: Ayman J. Oweida, Zeinab Sherifi, Yaoxian Xu, Siham Sabri, Bassam Abdulkarim. Evolving biological and clinical concepts of radiation delivery in NSCLC: response to ablative versus fractionated radiotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2014-865

Abstract A192: Role of O(6)-Methylguanine-DNA Methyltransferase in the Proliferation/Invasion Dichotomy and Differential Effect of FAK inhibitor Y11 based on expression of MGMT in Glioblastoma.

Introduction: Glioblastoma multiforme (GBM) is the most frequent and aggressive form of malignant primary brain tumors in adults. Prognosis for patinets diagnosed with GBM remains poor, with 90% of patients recurring within 2 years. Increased proliferation and invasion are the major hallmarks of GBM. Previous studies reported an inverse correlation between proliferation and invasion in GBM, a phenomenon termed the migration/proliferation dichotomy. Our laboratory has established for the first time the role of MGMT The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) as a potential negative regulator of GBM angiogenesis and invasion in GBM. SPARC (secreted protein acidic and rich in cysteine) is known for its pro-invasive and anti-proliferative effects in GBM. SPARC interacts with β1 integrin and activates focal adhesion kinase (FAK), a key regulator of migration and proliferation. FAK is overexpressed in patients diagnosed with GBM and has been proposed as a molecular target in GBM. Y11, a novel specific small molecule inhibitor highly selective for Tyr-397, the main autophosphorylation site of FAK has not been tested in GBM. Methods: We used U87/Empty vector (U87/EV) negative for MGMT and its counterpart overexpressing MGMT (U87/MGMT), T98/EV with constitutive expression of MGMT and its knockdown counterpart T98/MGMT-shRNA in addition to GBM cell lines with different levels of MGMT expression (U251, U138, LN18 and A172). The effect of Y11 was investigated using MTT proliferation assay (inhibiting concentration at 50%) and western blotting to assess p-FAK- Tyr-397, Total FAK and SPARC levels. Results: Using isogenic overexpression (U87/EV, U87MGMT) and knockdown (T98/EV, T98/shRNA) models of MGMT in GBM, we showed that expression of MGMT was associated with increased proliferation and decreased invasion. Western blotting (WB) analysis in isogenic cell lines for MGMT and other cell lines with different levels of MGMT expression (U251, U138, LN18 and A172) revealed a striking inverse relationship between expression of MGMT and SPARC. Importantly, we showed that treatment with the small molecule inhibitor Y11, which specifically inhibits FAK Tyr397 induced a dose dependent decrease of pFAK in all cell lines, while total FAK levels were unchanged. This decrease was accompanied by decreased proliferation in all cell lines. Conclusion: Our study reveals that MGMT regulates the proliferation/invasion dichotomy through regulation of SPARC and FAK and highlights the differential effect of Y11 on proliferation and invasion based on expression of MGMT. This will lead to the identification of new biomarkers of invasion and proliferation and prospective therapies targeting molecular effectors involved in invasion and proliferation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A192. Citation Format: Kenny Chatoor, Siham Sabri, Bassam Abdulkarim. Role of O(6)-Methylguanine-DNA Methyltransferase in the Proliferation/Invasion Dichotomy and Differential Effect of FAK inhibitor Y11 based on expression of MGMT in Glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A192.

Abstract C2: Cross-talk between the O(6)-Methylguanine-DNA methyltransferase (MGMT) and p53 in glioblastoma multiforme.

Background. Glioblastoma multiforme (GBM) is a fatal malignant primary brain tumor in adults with a median survival time of approximately 14.6 months, despite treatment with surgery, concurrent radiation therapy (RT) and the alkylating agent temozolomide (TMZ). O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein involved in resistance to TMZ in GBM. Our group showed that MGMT is a critical regulator of GBM angiogenesis, invasion and response to antiangiogenic treatment (sunitinib). While sunitinib treatment significantly decreased invasion in MGMT-positive cell lines, it led to a significant increase of invasion in MGMT-negative cell lines. Our group launched the first phase II clinical trial of concurrent sunitinib with RT and TMZ for selected patients with MGMT-positive tumors. The p53 tumor suppressor protein is involved in proliferation, angiogenesis and response to RT. Previous studies showed the effect of p53 in regulating MGMT levels. The role of MGMT and p53 in response to RT and sunitinib is unknown. We hypothesize that MGMT may affect levels of p53 and response to RT and sunitinib treatment. Results. We used isogenic human GBM cell lines isogenic for MGMT and assessed p53 levels by Western blotting and immunofluorescence. We showed for the first time that stable transfection of GBM cell line negative for MGMT (U87MG) and harboring wild type TP53 with a vector encoding for MGMT (U87MGMT) led to significant increase of p53 expression and its nuclear localization. Strikingly, increased p53 in U87MGMT cells did not lead to increased p21 protein, one of the main targets of p53. As shown by western blotting analysis, ionizing radiation (6 and 10 Gy) did not increase p21 levels compared to sham control. Interestingly, it was previously shown that irradiation does not lead to p21 activation in GBM cells with basal overexpression of p21 mRNA and can even lead to its repression as a mechanism of resistance to RT. We suggest that MGMT could be an upstream regulator of this resistance. Conclusion. Our study revealed a novel mechanism for regulation of p53 through MGMT, which may affect response to RT in GBM. We will further establish the relationship between MGMT/p53 status and response to combined RT and sunitinib in GBM cell lines and samples from patients enrolled in our clinical trial. This will lead to more efficient strategies of treatment for patients with tumors unresponsive to combined RT and sunitinib. Grant acknowledgments. This work has been funded by the Cedar Cancer Foundation, Research Institute of McGill University Health Center and the McGill-CIHR Drug Development Training Program (DDTP). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C2. Citation Format: Mariia Patyka, Bassam Abdulkarim, Yaoxian Xu, Siham Sabri. Cross-talk between the O(6)-Methylguanine-DNA methyltransferase (MGMT) and p53 in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C2.

Abstract 2493: Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion.

Background: Glioblastoma multiforme (GBM) is the most frequent and most aggressive form of primary malignant brain tumors in adults. The dismal prognosis of GBM patients stems from the highly angiogenic and invasive behavior of GBM tumor cells. O 6 -methylguanine-DNA methyltransferase (MGMT), a DNA repair protein ubiquitously expressed in normal tissues has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. We reported for the first time an inverse relationship between expression of MGMT and the angiogenic and invasive profile of GBM cell lines. The mechanisms by which MGMT affects angiogenesis and invasion are unknown. We hypothesized that interactions of MGMT with binding proteins (BPs) may account for additional functions beyond its known role as a DNA repair protein. Methods: As a first screening to identify MGMT-BPs with a functional relevance for invasion and angiogenesis, we performed affinity purification of MGMT-BPs following overexpression of FLAG-tagged MGMT and mass spectrometry analysis using 293T-Flag/MGMT and control Flag-tagged empty vector (293T-Flag/EV). Lysates were subjected to affinity purification using an anti-Flag monoclonal antibody covalently attached to agarose resin. The affinity bound FLAG fusion proteins were eluted and separated on SDS-PAGE. Coomassie Blue staining enabled the identification of 6 bands including Flag-MGMT in 293T-Flag/MGMT but not the Flag/EV control. The bands were excised from the gel, subjected to trypsin digestion and identified by liquid chromatography-tandem mass spectrometry. The resultant MS/MS spectra were searched against a proteome database for peptide matching and protein identification. Proteins were identified with high confidence using Scaffold software. Results: Our analysis provided evidence for binding of MGMT to 120 BPs. Using gene ontology (GO) database to search for functional categories, we identified proteins involved in DNA repair, ubiquitin pathway, DNA replication and transcription, RNA metabolism and processing, cell cycle and division, response to stress and cell death. Importantly, we identified proteins involved in cell motility and/or angiogenesis, cytoskeletal-related proteins (15 proteins), small GTPases family and their regulators (10 proteins, such as Rho guanine nucleotide exchange factor 2) and two proteins involved in angiogenesis (Endoribonuclease Dicer and Ribonuclease inhibitor). We also used T98G a human GBM cell line with constitutive expression of MGMT to perform immunoprecipitation of endogenous MGMT (anti-MGMT antibody or the IgG1 isotype control). Mass spectrometry and proteomic analysis of MGMT-BPs in T98G is underway. Conclusion: Our data provide new structural aspects of MGMT and shed light into the multifaceted role of MGMT, which may lead to the identification of novel therapeutic targets in GBM. Citation Format: Siham Sabri, Yaoxian Xu, Nicolas Stifani, Bassam S. Abdulkarim. Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion. [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 2493. doi:10.1158/1538-7445.AM2013-2493