Download Develop a reliable method to measure the phosphorylation status of

2016-2017 Novel Clinical and Translational Methods (NCTM)
Pilot Program Application
Name(s): _Isabel R. Schlaepfer, PhD_______________________________________
Department(s) and School/College: ____SOM/ DOM/ Medical Oncology____________
Project Title: Develop a reliable method to measure the phosphorylation status of
phosphatidylinositol molecules in cells, tumors and body fluids.
Applying for:
• Phase I (Identify Novel Methodological Development Need)
• Phase II (Novel Method Development Plan)
This application qualifies as:
• Underserved and Minority Investigator
• Female Principal Investigator
Isabel R. Schlaepfer, Ph.D. | Assistant Professor
University of Colorado School of Medicine | Division of Medical Oncology
Genitourinary Cancer Program MS 8117 | 12801 E. 17th Ave, Room L18-8101D
Aurora, CO 80045 ph: 303-724-8867
[email protected]
Page 8
Title: Develop a reliable method to measure the phosphorylation status of phosphatidylinositol
molecules in cells, tumors and body fluids.
The goal of this proposal is to develop an easily accessible method for the extraction, detection and quantitation
of phosphatidylinositol phosphates (PIPs), with different degrees of phosphorylation, from cells, tissues and
fluids. Ideally, the technique developed will be available to any investigator in the University of Colorado, either
in their own laboratories or through collaboration, will not involve the use of radioactive or hazardous reagents
and will have a reasonable cost per sample. It should allow identification and quantitation of molecular species
of PIPs, including the acyl chains, the number and, ideally, the position of the phosphate groups on the inositol
ring.
Specifically, I am interested in measuring accurately the PIPs that are implicated in AKT activation, as part of my
investigations in prostate cancer. But the potential interest of such a technique far exceeds the scope of cancer
research. AKT phosphorylates upwards of 200 target proteins that regulate gene expression, protein synthesis,
cell cycle progression, cytoskeleton organization, and cell metabolism, PIPs regulate many other proteins
involved in signaling. The availability of a reliable technique to quantitate PIPs could potentially answer important
questions, for example: 1- how diseases such as cancer, cardiovascular disease, obesity or diabetes are
established and progress, 2- how cells and tissues are involved at different stages of disease, 3- which types of
cellular stress factors trigger signaling leading to adverse cellular responses, 4- how the use of inhibitors or
genetic manipulation could interfere with PIP/AKT signaling, or 5- how effective available treatments are in
blocking signaling pathways. Information can be obtained from many experimental settings, ranging from cells
in culture to animal models of disease and samples from human patients.
Rationale & Background:
Phosphatidylinositol-3-kinase (PI3K) activity is known to play crucial roles in signaling events relevant to a wide
number of cells and tissues, which in turn affect some of the most common diseases in humans, including cancer,
obesity or diabetes. In fact, inhibitors of this enzymatic activity are currently undergoing clinical trials to study
their efficacy in treating cancer.
PI3K isoforms catalyze the incorporation of phosphate in the position 3 of the inositol ring of phosphatidylinositol
(PI), phosphatidylinositol monophosphate (PIP) or phosphatidylinositol bisphosphate (PIP2). The levels of these
lipids affect intracellular signaling through their binding to proteins containing specific domains such as pleckstrin
homology (PH), phox (PX) or FYVE domains. One example of this signaling is the binding of PI(3,4,5)P3 to the
PH domain of Akt, which is then translocated to the plasma membrane and phosphorylated to initiate a signaling
cascade which affects cell proliferation, survival and metabolism. Most scientist measure the activation of AKT
via western blotting, using antibodies that target phosphorylated forms of AKT. This is a valid method but it is
indirect, since the initial steps of the activation occur at the plasma membrane, where the phosphatidylinositols
become modified in response to growth factors or other stimuli.PIPs are modified in two major ways: via
phosphorylation by PI kinases and, less known, via PIP phosphatases like INPP5K and PTEN. The INPP5K
gene codes for a phosphatase that removes the 5 phosphate group from each of the 3’, 4’, and 5’ positions of
PI(3,4,5)P3 and related phosphoinositides. Considering the key role that the PI3K/AKT pathway plays in cancer,
obesity and diabetes, changes in the activities of these PIP phosphatases has a tremendous impact. For
example, insulin-induced intracellular production of PI(3,4,5)P3 and phosphorylation of AKT decreased when
INPP5K was overexpressed, but increased when endogenous levels of INPP5K were reduced by means of
antisense oligonucleotide in ovary cells overexpressing insulin receptors.
The phosphatase INPP5K is the focus of my investigations in prostate cancer. I have found that modifying lipid
metabolism via CPT1A in the mitochondria produces significant changes in phosphatidylinositol content and
decreased activation of AKT. However, I cannot measure accurately the specific PIP species involved (Figure
1). Currently, it is not possible with the current methods available to quantify the type of PIP isomers. This is very
important since different PIPs, (PIP2S and PIP3S) can bind PH, PX or FYVE domains on many proteins with
different specificities.
Figure 1: Diagram of the site of action of INPP5K and
its potential effect on androgen receptor (AR)
expression via decreased activation of AKT. The
decreased expression of CPT1A (1) leads to increased
phospholipid degradation and increased INPP5K
phosphatase activity, resulting in decreased AKT
expression and activation (2). This consequent
decrease in p-AKT leads to increased AR expression (3)
and action, increasing PSA (KLK3) and FASN and
resulting in a more differentiated, lipid-loaded
phenotype. PROBLEM: How can I measure the
molecular forms of PIPs that are substrates and
products of phosphatase actions?
Only a fraction of the big number of studies published on PI3K include actual measurements of the levels of the
substrates and products of these enzymes. This fact is mainly due to the scarcity of reliable analytical methods
available to most investigators. Analysis of these lipids is complicated by several factors including extraction
from tissues, detection, identification and quantitation. Extraction is difficult because the varying numbers of
phosphate groups result in different partitioning rates in the solvents traditionally used for extraction. As for
identification, many different molecular species exist based on the number of phosphate groups and their
positions as well as the acyl chains esterified to the glycerol backbone, which can vary greatly in length (typically
between 14 and 22 carbons), degree of unsaturation, position and configuration of the double bonds. Detection
and quantitation are further complicated by the great differences in levels of the different molecular species,
which span several orders of magnitude and require an analytical method with an adequate dynamic range.
Perhaps the most common method of measuring PIPs has consisted in radiolabeling using myo-inositol or
inorganic phosphate. In most cases, this is followed by hydrolysis and analysis of the polar group using
chromatographic separation. In addition to the cost, regulatory constraints and potential risks of using radioactive
materials, this method does not reveal the acyl chain composition of the PIPs. The same problem affects the
use of antibody-based EIA methods, which are also affected by the potential promiscuity of the antibodies used.
Because of these caveats, most of the recent efforts in measuring PIPs have used mass spectrometry as a
detection and identification technique. One promising approach involved the methylation of the phosphate
groups in PIP3 to overcome the extraction problems mentioned above. However, this approach requires the use
of (trimethylsylil) diazomethane, a reagent that is toxic and potentially hazardous, which probably explains why
this method has not been widely adopted in the PI3K scientific community. Alternatively, a method used within
the LipidMAPS consortium based on the analysis of intact PIP molecules has also failed to become adopted
because of concerns about sensitivity and reproducibility of extraction.
This proposal aims to recruit one or more collaborating groups within the CCTSI network to explore new
approaches to determine the levels of these lipids. Most likely, a successful new method will build on experience
gleaned on the techniques mentioned above and improve particularly on the extraction and chromatographic
separation of PIPs prior to mass spectrometry-based analysis. It is no doubt a challenging project, but one that
could yield an enormous benefit for many researchers, within the University of Colorado and the wider scientific
community, interested in the signaling mechanisms involved human disease. Furthermore, this novel method
will allow investigators to reliably quantitate PIPs from cells, blood, urine or tissue biopsies from different
pathologies or drug treatments.