Academic Background

PTEN phosphatase and tumor suppressor, CNS Stem Cells and neural tumors.

Even though phosphorylation of phosphatidylinositols by phosphoinositide 3-kinase has an important and pervasive role in the nervous system, little is known about the phosphatases that reverse this reaction. Recently, such a phosphatase, PTEN, was cloned as a tumor suppressor for gliomas. We now know that PTEN is a tumor suppressor for many tumor types and is a phosphatidylinositol phosphatase specific for the 3-position of the inositol ring. PTEN is expressed in most, if not all, neurons and is localized in the nucleus and cytoplasm.   

Figure 1. Balance between phosphatidylinositol 4,5 bisphosphate and phosphatidylinositol 3,4,5 trisphosphate is determined by relative activities of phosphatidylinositol 3-kinase and PTEN phosphatase.

Figure 2. Micrograph showing that exogenously expressed green fluorescent protein PTEN is present in both the nucleus and cytoplasm of HeLa cells.

Figure 3. Fit of kinetic data for activation of PTEN by phosphatidylinositol 4,5 bisphosphate.

Figure 4. Magnetic resonance image of patient's brain bearing a large glioma brain tumor.

We are examining the regulation of PTEN activity at the protein level. In recent experiments, we measured reaction rates for varying concentrations of monodisperse (i.e., not in a vesicle or micelle) PI(3,4,5)P₃. The kinetic curves did not follow the typical Michaelis-Menten form, especially at higher PI(3,4,5)P₃ levels. The kinetic curves were sigmoidal, indicating that the enzymatic activity increases as the reaction progresses. One possible explanation is that the PTEN product, PI(4,5)P₂, is a positive regulator of PTEN activity. We measured PTEN activity as a function of PI(4,5)P₂ concentration and found that PI(4,5)P₂ activated PTEN with a K_act = 20 micromolar. This regulation is specific. For example, PI(3,4)P₂ and PI(3,5)P₂ do not activate PTEN. Based on these data, we propose that PI(4,5)P₂ binds to a site distinct from the phosphatase active site, induces an allosteric conformational change, and, thereby, activates PTEN, leading to a positive feedback loop for PTEN activity. This model predicts that PTEN would be preferentially activated at the PI(4,5)P₂-bearing plasma membrane or at PI(4,5)P₂ -rich membrane domains.

We also are carrying out two projects to improve therapies for brain tumors. Glioblastoma multiformi is the most aggressive brain tumor, and despite treatment with surgery, radiotherapy and chemotherapy, the prognosis is poor. Some cells are resistant to these therapies, and eventually the tumor recurs. Recent models suggest that the resistant cells are cancer stem cells. These cells express stem cell markers, self-renew and can differentiate along developmental lineages. Cancer therapies directed against the cancer stem cells might yield real cures.

A problem in this field is that cancer cells change in culture. As a result, it is important to use freshly excised tumors or low passage cell cultures. We are collaborating with Dr. Rick Moser of the Neurosurgery Department. We have put a series of these tumors into culture with defined medium. Under these conditions, the cells grow as aggregates of cells, known as neurospheres. Cells grown under these conditions more closely resemble the original tumor cells.

We are testing a new approach to enhance the efficacy of temozolomide (TMZ), which is the chemotherapy drug of choice for brain tumors. In particular, we are modulating signaling pathways to enhance TMZ-induced cell death and senescence of cancer stem cells. We have observed enhanced efficacy both in culture and in mice with ex vivo treated cells.

We also are studying meningiomas, which are common brain tumors and usually benign. Resection of meningiomas can be difficult for the surgeon and the patient. Drugs to reduce tumor growth and prevent tumor recurrence would be a helpful alternative, but unfortunately, chemotherapy has not been useful for these tumors. As a result, we wish to develop therapies directed to specific signaling pathways that drive proliferation of meningioma cells. Using protein arrays, we have found that 15/15 meningioma cultures have strongly activated epidermal growth factor receptor, and 14/15 cultures have activated platelet-derived growth factor receptor. Drugs that inhibit these receptors slow meningioma cell proliferation and induce cell death.

We hope to carry both the glioblastoma and meningioma projects to clinical trials