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Studies on the mechanism of action of
Ras in human glioblastoma:
new approaches to brain tumor therapy
utilizing the Ras-inhibitor Salirasib
My PhD studies at Prof. Yoel Kloog's lab focused on the concept of Salirasib as a unique therapeutic drug that may protect against malignant glioblastoma.
When I joined the lab it was obvious that Salirasib can efficiently induce dislodgement of active Ras from cellular membrane and thus reduce level of cellular active-Ras. It was shown that treatment with Salirasib led to cell growth inhibition of several cell lines transformed by constitutively activate Ras, including N-Ras-transformed human melanoma cells, human skin cancer cells (merkel cell carcinoma, MCC) that depended on signaling of tyrosine kinase receptors and LNCaP and CWR-R1 prostate cancer cells that associated with upregulation of growth factor mediated pathways.However, the cellular mechanisms mediating Salirasib's efficacy remain unrevealed. To deepen our understanding regarding Salirasib's mechanism of action in cells transformed by constitutively activate Ras, I opted to conduct my studies in human glioblastoma cells that harbor high level of chronically active WT-Ras due to their highly activated tyrosine kinase receptors.
I employed genome-wide gene expression profiling along with classical biochemical techniques to identify the main core of transcriptional regulation that governs the cellular response to Salirasib in glibolastoma cells. In a collaborative study with the laboratory of Prof. Gideon Rechavi from Sheba Medical Center, I discovered a phenomenal gene expression reduction in a number of prominent hypoxia induced factor (HIF-1α) target genes as result of Salirasib administration. Treatment with the drug reduced the expression of numerous glycolysis enzymes that are positively regulated by HIF-1α, leading to glycolysis shutdown and to severe drop in ATP levels (published in Cancer Research).
To obtain a global dissection of the transcriptional response to Salirasib, a follow-up collaborative study with Dr. Rani Elkon and Prof. Ron Shamir, was carried to discover additional transcription factors that participate in the transcriptional response to Salirasib treatment. I identified E2F1 to be the predominant Ras-dependent component associated with cell-cycle arrest. Evidently, inhibition of Ras by Salirasib promoted decreased expression of key E2F1-target genes that are critical for cell-cycle progression (published in International Journal of Cancer).
In the scope of a later work I focused on cell death events (apoptosis) that are induced in gliblastoma cells following Salirasib treatment. My findings revealed that Salirasib severely inhibited transcription of a major anti-apoptotic regulator, Survivin, and concomitantly activated the apoptotic machinery. These studies indicated that glioblastoma's resistance to apoptosis can be canceled by a single Ras inhibitor which targets both, Survivin, a critical anti-apoptotic regulator, and the intrinsic mitochondrial apoptotic machinery (published in Molecular Cancer Therapeutics).
Complementary research in collaboration with Prof. Ronit Pinkas-Kramarski, has demonstrated Salirasib's anti-apoptotic effects in cancerous prostate cell lines (published in Biochemical Pharmacology). Altogether, these discoveries provided the first explanation of cell death mediated by Ras inhibition through downregulation of the anti-apoptotic machinery. These results showed that Salirasib can induce critical metabolic change as well as cytostatic effect in human glioblastoma cells. Thus, I have suggested that targeting oncogenic Ras with Salirasib may replace the need of combined inhibition of both, HIF-1α and E2F1, two distinct “addictive oncogenes” of the glioblastoma cells (review article published in Drug Resistance Updates).
In light of the complexity of Ras pathways and their consequent downregulation, I sought to decipher the core molecular response to Ras inhibition in five cancerous cell lines. By employing gene-expression profiling I identified a distinctive transcriptional response to Salirasib that was common to all tested cancerous cell lines and yet not shared by normal WT-fibroblast. These studies provided a strong support to the conclusion that Salirasib specifically re-regulates defective Ras pathways in human tumor cells. The results (published in Cancer Research), suggested that patients with cancerous tumors over-expressing activated Ras pathways, could be benefit by a treatment with Salirasib. This study provided the first gene expression data of tumors cells react to treatment with Salirasib and the identified transcriptional response has become a potential guideline for assessing the drug administration impact in ongoing clinical studies in human patients.
In light of my thorough studies with the drug, I summarized the beneficial aspects of Salirasib as anti-cancerous drug and have suggested that it should become an influential therapeutic tool for treatment of human malignancies that harbor high activated Ras levels (review article published in Recent Patents on Anti-Cancer Drug Discovery).
Lastly, due to the significant impact of Salirasib treatment on metabolic pathways of tumorigenic cells, such as glioblastoma and pancreatic tumor cells, we opted to focus on the recent discoveries related to metabolic mechanisms of pancreatic tumor cells. We summarized the latest findings in the field including the beneficiary effects of several pre-clinical drugs in a review article published in Cell Death and Disease.