Faris Farassati

Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR−/− (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

Ral overactivation in malignant peripheral nerve sheath tumors.

ABSTRACT Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.

Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Δ) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Δ cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Δ cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Δ cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, β-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Δ cells with a decrease in G1 and increase in S and G2 fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Δ cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. [Mol Cancer Ther 2008;7(11):3586–97]

Oncolytic viruses and cancer therapy

In the hopes that a better understanding of cancer biology would allow for the development of novel, more effective therapeutics, a concerted effort over the past 20 years has focussed on the molecular mechanisms of cell growth control and tumorigenesis. Recently, there has been a renewal of interest and optimism in the field, on account of a confluence of ideas from molecular oncology and virology, leading to the development of novel virus-based therapeutics for the treatment of cancer. Viruses have been used in the past as potential cancer therapeutics; a large variety of agents have been tested, including rabies, adenovirus, paramyxovirus, Newcastle disease virus and mumps virus [1]. Although ‘responses’ have been documented, little has emerged from such trials in terms of a longstanding, oncolytic virus therapeutic mainstay. Fortunately, new technologies and insight have allowed for a re-examination of oncolytic viruses; an understanding of cancer biology and signalling, for example, has permitted the characterisation of the selectivity of viruses such as reovirus for cells with an activated Ras signalling pathway [2,3]. Discovery of the ability of adenovirus E1B protein to interact with the tumor suppressor, p53, not only helped to delineate p53’s role in cell cycle control, but also provided the foundation for the development of E1B-deleted mutants as potential cancer therapeutics [4,5]. Similarly, an awareness of herpes virus biology has permitted the engineering of non-neurovirulent mutants lacking genes necessary for * Corresponding author. Tel.: +1-403-2207548; fax: +1-4032708520. E-mail address: plee@ucalgary.ca (P.W.K. Lee).

Utilizing Ras Signaling Pathway to Direct Selective Replication of Herpes Simplex Virus-1

Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication) is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway). This mutant HSV-1 was named as Signal-Smart 1 (SS1). A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05), while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a “prototype” for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

Ras signaling influences permissiveness of malignant peripheral nerve sheath tumor cells to oncolytic herpes.

Lack of expression of neurofibromin in neurofibromatosis 1 and its lethal derivative, malignant peripheral nerve sheath tumors (MPNSTs), is thought to result in the overactivation of the Ras signaling pathway. Our previous studies have shown that cells with overactivation in the Ras pathway are more permissive to infection with herpes simplex virus 1 and its mutant version R3616. In this study, we show that among five different mouse MPNST cell lines, only the ones with elevated levels of Ras signaling are highly permissive to infection with oncolytic herpes G207. Specific inhibitors of the Ras, ERK, and JNK pathways all reduced the synthesis of viral proteins in MPNST cells. The cell lines that contained lower levels of Ras and decreased activation of downstream signaling components underwent an enhancement in apoptosis upon exposure to G207. Additionally, mouse SW10 Schwann cells were able to become infected by parental herpes but were found to be resistant to G207. The immortalization of these cell lines with the expression of SV40 large T antigen increased the levels of Ras activation and permissiveness to oncolytic herpes. A Ras/Raf kinase inhibitor reduced the synthesis of both herpes simplex virus-1 and G207 proteins in SW10 cells. The results of this study, therefore, introduce Ras signaling as a divergent turning point for the response of MPNST cells to an assault by oncolytic herpes.

Inhibition of Mesothelin as a Novel Strategy for Targeting Cancer Cells

Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA) decreased viability of cancer cells from different origins such as mesothelioma (H2373), ovarian cancer (Skov3 and Ovcar-5) and pancreatic cancer (Miapaca2 and Panc-1). Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of β-Catenin, an important marker of EMT (epithelial-mesenchymal transition). Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress) pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429) with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.

Protein kinase C-β inhibitor enzastaurin (LY317615.HCI) enhances radiation control of murine breast cancer in an orthotopic model of bone metastasis

SummaryRadiation therapy is a widely used treatment for metastatic bone cancer, but the rapid onset of tumor radioresistance is a major problem. We investigated the radiosensitizing effect of enzastaurin, a protein kinase Cβ (PKCβ) inhibitor, on bone tumor growth and tumor-related pain. We found that enzastaurin enhanced the effect of ionizing radiation on cultured murine 4T1 breast cancer and murine endothelial cells, suppressing their proliferation and colony formation. Enzastaurin and ionizing radiation also induced caspase-mediated apoptosis of 4T1 cells to a greater degree than radiation alone. Enzastaurin treatment of 4T1 cells blocked the phosphorylation of PKCβ, as well as Ras and two of its downstream effectors ERK1/2 and RAL-GTP. Using an orthotopic model of bone metastasis, we observed that a combination of enzastaurin and localized radiation treatment reduced tumor blood vessel density, bone destruction and pain compared to single modality treatment. In conclusion, we demonstrate that inhibition of PKCβ in combination with localized radiation treatment suppresses tumor growth and alleviates pain as compared to radiation-only treatment. We also show that the radiosensitizing effect of enzastaurin is associated with suppression of tumor cell proliferation and tumor-induced angiogenesis possibly through inhibition of the Ras pathway.

Colorectal Cancer in Iran: Molecular Epidemiology and Screening Strategies

Purpose. The increasing incidence of colorectal cancer (CRC) in the past three decades in Iran has made it a major public health burden. This study aimed to report its epidemiologic features, molecular genetic aspects, survival, heredity, and screening pattern in Iran. Methods. A comprehensive literature review was conducted to identify the relevant published articles. We used medical subject headings, including colorectal cancer, molecular genetics, KRAS and BRAF mutations, screening, survival, epidemiologic study, and Iran. Results. Age standardized incidence rate of Iranian CRCs was 11.6 and 10.5 for men and women, respectively. Overall five-year survival rate was 41%, and the proportion of CRC among the younger age group was higher than that of western countries. Depending on ethnicity, geographical region, dietary, and genetic predisposition, mutation genes were considerably diverse and distinct among CRCs across Iran. The high occurrence of CRC in records of relatives of CRC patients showed that family history of CRC was more common among young CRCs. Conclusion. Appropriate screening strategies for CRC which is amenable to early detection through screening, especially in relatives of CRCs, should be considered as the first step in CRC screening programs.

Effect of sequential docetaxel followed by mTOR inhibitor temsirolimus on suppression of PI3K overactivation resistance mechanism.

88 Background: Mammalian target of rapamycin (mTOR) is a downstream regulatory protein of the PI3K/Akt signal transduction pathway. This is a common pathway for a several cell surface receptors including IGFR (Insulin-like Growth Factor Receptor) and EGFR (Epidermal Growth Factor Receptor). The activation of these receptors through PI3K/Akt pathway is essential in cell proliferation, angiogenesis, and anti-apoptosis process. The upregulation of PI3K could be a mechanism of resistance of mTOR inhibitors. Docetaxel (D) is commonly used in the treatment of lung cancer. We demonstrated previously that the sequence of D followed by mTOR inhibitor temsirolimus (T) in lung cancer cell lines (LCCL) had synergistic effect in suppressing cell proliferation compared with T→D. The exact mechanism of this effect is unknown. We studied the expression of mTOR and PI3K in these cell lines treated in different time points to investigate the activity of this pathway when using these sequences of drug treatment. METHODS Adenocarcinoma LCCL H2122 and H1437 were plated and exposed to temsirolimus 1000nM and docetaxel 100nM. The cell viability was measured by optical density (OD) at 24, 48, and 72h. We tested effect of drugs D and T alone as well as the sequence of D treated for 24h followed by addition of T and the reverse in both LCCL. We then prepared cell lysate at 24h, 48h, and 72h time points and studied the expression of phospho mTOR (pmTOR) and PI3K by western blot using antibody obtained from cell signaling. RESULTS The use of T alone increased the expression of PI3K in both H2122 and H1437 cell lines at 48h time point. The use of D had a variable response: absent in H1437 and present in H2122 at 48 H. The sequence of D→T suppressed the expression of pmTOR and PI3K at 48 and 72 h compared with the opposite sequence of T→D. CONCLUSIONS The combination of D → T is synergistic in suppression of pmTOR and inhibited the overactivation of upstream PI3K in both LCCL compared with opposite sequence. Therefore, the sequential treatment of D followed by T is able to overcome the PI3K overactivation mechanism of resistance in lung cancer cell line when treated with T. This would have implications in the use of these agents in treatment of lung cancer.