A Presentation on PTEN, the mTOR pathway, and Glioblastoma

 This presentation will cover an overview of many of the primary subjects of experimentation in the lab, including PTEN, the mTOR pathway, Glioblastoma, and the mechanism through which these are able to be studied: neuronal stem cell differentiation.

Background Information on Primary Topics of Experimentation

The mammalian target of rapamycin (mTOR) pathway responds to both intracellular and extracellular signals and functions as a regulator of cell metabolism, proliferation, and growth. The mTOR protein itself belongs to the PI3K protein kinase family and nucleates two signaling complexes: mTORC1 and mTORC2. p53, a common cell cycle regulator, inhibits mTORC1 signaling by increasing the transcription of PTEN, which is a negative regulator of the mTOR pathway. Human PTEN is a 403–amino acid protein localized to the nucleus that contains four domains and implicates cellular processes associated with cell polarity, growth, metabolism, cell cycle, and survival. MAP1B of the microtubule-associated protein family is another significant focus of experimentation. This protein encodes for a precursor protein and has a role in structural molecule activity and microtubule binding. When phosphorylated, MAP1B plays a role in the progression of cytoskeletal changes that correlate with neurite expansion and growth.

NPCs or neural precursor cells are the combined set of both neural progenitor cells and neural stem cells derived from embryonic stem cells and induced pluripotent stem cells capable of differentiation into both neuronal and glial cells. Fetal NPCs are specifically derived from the embryo and are extracted from tissue and purified before being used in experimentation. In order to confirm fetal cells to be NPCs, immunofluorescence (IF) staining, a process in which fluorescently labeled antibodies are used to target specific antigens, is utilized. H1s, H7s, H9s, H13s, and H14s are among the first five derived lines of human embryonic stem cell lines or hESCs. A novel method of long-term human embryonic stem cell maintenance titled small molecule inhibition uses a unique combination of small molecule inhibitors chosen through research of the stem cell line and replaces the use of feeders and allows us to maintain cells through the simpler method of single cell passaging.

Glioblastoma (GBM) is a form of malignant cancer in the brain or spinal cord that currently has no cure and only symptomatic treatment and is localized to the central nervous system. Glioblastoma is quite unique in its ability to vascularize and thus the tumor self-sustains in a manner that makes tumor growth difficult to repress and increases chances of post-radiation recurrence. Radiation therapy may induce the conversion of glioblastoma stem cells (GSCs) to epithelial cells and pericytes that promote tumor growth and recurrence. GBM lines from LN2 can further be utilized to study MEK inhibition. MEK inhibitors are selective for the mitogen-activated protein kinase enzymes MEK1 and/or MEK2 and thus have clinical significance in cancer treatments.

Current Experimentation

In the past 10 weeks, the focus of lab work has been on maintaining and developing stem cells for future experimentation on the role of MAP1B as a downstream effector of GS3KB-mediated resistance to rapamycin. Rapamycin is an immunosuppressant drug that inhibits the mTOR pathway. Fetal neural precursor cells were procured from Week 22 human embryonic tissue and immunofluorescence staining was conducted for MAP1B and phosphorylated MAP1B. hESCs of the H9 cell line were differentiated into neurons through the protocol of StemCell Technologies. This involves the preparation of a differentiation medium, preparation of PLO/Lamnin cell culturing vessels, preparation of maturation medium, rosette selection, and cell passaging/switching of the medium. Further immunofluorescence staining was conducted in order to confirm the identity of the fetal cells to be neural precursor cells. These fetal neural precursor cells were then plated to begin the differentiation process into neurons. The more effective and novel method of small molecule inhibition was used for the differentiation protocol. The specific small molecule inhibitors were selected based on previous research of the stem cell line. A new neuronal differentiation protocol was designed, one that was optimized for human neural precursor cells rather than previous differentiation protocols based on other animal models. A change in morphology occurred in the fetal cells. Thus immunofluorescence was completed again to confirm fetal NPC identity. Glioblastoma lines were procured from LN2 and were grown and maintained for the investigation of MEK inhibitors.

Future Research

As the stem cell differentiation process completes, there will be more opportunity to analyze the role of MAP1B as a downstream effector of GS3KB-mediated resistance to rapamycin. Rapamycin is a known inhibitor of TORC1 but has limited use for GBM when used in chronic treatment. MAP1B, a mediator of this resistance, is predominantly expressed in early nervous system development but MAP1B regulation continues throughout adult stages of life. For this purpose, it may be valuable to apply in vitro conclusions learned in this experiment to in vivo experiments utilizing animal models: specifically the use of MAP1B KO mice. The effects of Rapamycin on MAP1B KO mice and MAP1B control mice can be observed. An in vivo experiment would produce more clinically significant results and allow the observation of lifelong MAP1B function. The addition of Epothilone D in conjunction with Rapamycin would further extend the clinical significance. Resistance to chronic rapamycin treatment occurs through induction of microtubular stability and Epothilone D promotes microtubular stability. The combination of Rapamycin and Epothilone D may result in a decrease of the anti-proliferative effects of Rapamycin and improve our understanding of the mechanism of resistance to drug therapies.