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Molecular Pathways
Insulin Receptor Substrate Regulation of Phosphoinositol-3 Kinase
Heather E. Metz and A. McGarry Houghton
Departments of Medicine and Molecular Pathology, University of Pittsburgh Cancer
Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
Correspondence:
A. McGarry Houghton, MD
University of Pittsburgh School of Medicine
NW628 Montefiore
3459 Fifth Avenue
Pittsburgh, PA 15213
[email protected]
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Abstract
Insulin receptor substrates serve as downstream messengers from activated cell surface
receptors to numerous signaling pathway cascades.
One of these pathways,
phosphoinositol-3 kinase (PI3K), frequently displays aberrant function in the setting of
cancer. IRS proteins are capable of both regulating and activating PI3K, depending on the
cell of origin. As such, both pro-host and pro-tumor functions have been described for IRS
proteins in human cancers. IRS proteins may eventually serve as biomarkers of PI3K
activity, and serve a much-needed role as a guide to using targeted pathway therapy.
Additionally, IRS-1 could be indirectly targeted in lung cancer, by inhibiting neutrophil
elastase, which functions to degrade IRS-1 in lung tumor cells, thereby generating PI3K
hyperactivity.
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Background
Insulin receptor substrates (IRS) are signaling adaptor proteins that function as
intermediates of activated cell surface receptors, most notably for the insulin receptor (IR)
and insulin-like growth factor receptor (IGF-IR) (1-3). More recently, IRS proteins have
been shown to signal downstream of integrin, cytokine, and steroid hormones receptors as
well (4, 5), although these functions are poorly understood when compared to the
“canonical” (IR and IGF-IR mediated) properties of IRS proteins. By mediating the activities
of these receptors, IRS proteins interface with several signaling pathways thereby
impacting numerous aspects of cell behavior including metabolism, motility, survival, and
proliferation (Figure 1). The majority of IRS protein research to date has centered upon
the study of glucose metabolism and the pathogenesis of diabetes. Reports pertaining to
the roles of IRS proteins in cancer progression are beginning to emerge (6).
Six IRS proteins have been described, however; IRS-3 is expressed only in rodents
(7); IRS-4 displays limited tissue expression (brain and thymus) (8); and IRSs-5 and -6 are
structurally dissimilar from the others (9). Therefore, most of the attention has been
focused on IRS-1 and IRS-2, both of which are widely expressed. IRS proteins share similar
structural domains including an N-terminal pleckstrin homology (PH) domain and a
phospho-tyrosine binding (PTB) domain, which is required for binding NPEY motifs in the
juxtamembrane region of ligand-activated IR and IGF-IR (10). The carboxy terminus
contains numerous serine and tyrosine phosphorylation sites that bind PTB containing srchomology-2 (SH2) proteins including p85, Grb2, Nck, the phosphotyrosine phosphatase
SHP2, Fyn, and others (11). Although IRS proteins are not catalytically active, they are
capable of impacting numerous signaling cascades via interaction with SH2 proteins.
Despite sharing binding partners and structural similarities, IRS-1 and IRS-2
functions are not entirely overlapping. IRS-1-/- mice display low birth weight and glucose
intolerance, but do not develop overt diabetes (12). The generation of these mice led to the
discovery of IRS-2, which was believed to compensate for the loss of IRS-1 and prevent
additional metabolic derangements.
IRS-2-/- mice have subsequently been shown to
develop diabetes as a consequence of decreased ß-cell function and insulin resistance (13).
Therefore, IRS-1 and IRS-2 possess both overlapping and unique properties, although their
relative contribution to cancer growth and invasiveness has yet to be elucidated.
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Though IRS proteins signal through many pathways, their predominant function
appears to be activation and/or regulation of the phosphoinositol-3 kinase (PI3K) and
extracellular signal-regulated kinase (ERK) pathways. PI3K is a heterodimer with separate
regulatory (p85) and catalytic subunits (p110). In its resting state, PI3K exists as an
inactive p85—p110 complex. Upon the activation of a receptor tyrosine kinase (RTK),
meaning phosphorylation of its cytoplasmic tail, the p85-p110 complex is recruited to the
receptor by interaction of a SH2 domain on p85 with phosphotyrosine residues on the RTK
(14). This interaction is believed to release the inhibitory effects of p85 on the catalytic
p110 (15). p110 is now able to interact with its lipid substrates, the phosphatidylinositols,
and convert PIP2 to PIP3. Recruitment of PI3K by RTK also puts p110 in close proximity to
these lipid substrates residing in the plasma membrane. The major exception to this
schema is that PI3K can be activated by signal adapter proteins, such as IRS-1 and IRS-2,
rather than by RTKs themselves (5). Of note, IRS-mediated activation of PI3K requires that
phosphorylated YMXM motifs occupy both SH2 domains within p85 (16).
Generation of PIP3 by activated PI3K near the plasma membrane results in interaction
with, and subsequent phosphorylation of, its primary substrate, Akt (17). Once activated,
pAkt utilizes extensive downstream signaling pathways to enhance tumor viability in one
of three ways: cell survival, cell proliferation (number), and cell growth (size) (18). The
avoidance of apoptosis, achieved by the direct phosphorylation of BAD by pAkt, is generally
considered the predominant function of PI3K/Akt in cancer cells (19). However, PI3K/Akt
also promotes tumor cell proliferation by causing an accumulation of cyclin D1, which
regulates G1/S phase transition (20). This is accomplished by inhibition of p27, p21, and
glycogen synthase kinase-3β (GSK3β), which target cyclin D1 for proteasomal degradation,
when active (21, 22).
PI3K activity appears to be regulated in two ways. The first is simply the activation
of the p85 subunit, which maintains p110 in an inactive state at baseline. The second is the
constitutively active negative repressor, phosphatase and tensin homologue (PTEN). PTEN
regulates the output of PI3K by dephosphorylating PIP3 back to PIP2 (23). Mutation in
PTEN is relatively common in cancers, and has been linked to PI3K hyperactivity in several,
including prostate carcinoma, hepatocellular carcinoma, melanoma, renal-cell carcinoma,
and glioblastoma, among others (24-28). Interestingly, PTEN mutation is rare in some
cancers, including lung cancer (29). The common explanations for PI3K hyperactivity in the
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setting of preserved PTEN expression has been activation of PI3K by K-ras and genetic
mutations within the PI3K pathway (e.g. PIK3CA), both of which possess the ability to bypass regulatory machinery (30-32).
Although generally considered positive effectors of growth factor, IRS proteins may,
in fact, function as homeostatic regulators of PI3K output in certain tissues. Supporting
evidence of this theory includes the fact that IRS—p85 interaction takes place within the
cytosol, pulling PI3K away from its lipid substrates and creating a compartmentalization
phenomena for signaling (33). Furthermore, IRS proteins display low potency for ligand
interaction. As an example, IRS-1—Grb2 binding results in nearly ten-fold less pathway
output when compared to other common Grb2 binding partners (34).
IRS Proteins in Cancer
Pre-clinical studies have demonstrated both pro-tumor and pro-host functions for
IRS proteins in cancer.
IRS-1 over-expression has been shown to induce malignant
transformation in both embryonic mouse fibroblasts (35) and NIH3T3 fibroblasts (36, 37).
Such cells are capable of tumor formation in nude mice, a process displaying MEK/ERK
hyperactivity and requiring IGF-IR, as embryonic fibroblasts derived from IGF-IR-/- mice are
resistant to IRS-1 induced transformation (38). Oncogenic transformation by IRS-1 has not
been described for other cell types.
Both IRS-1 and IRS-2 are commonly over-expressed in hepatocellular carcinoma
(HCC), which is characterized by IR and IGF-IR signaling hyperactivity (39, 40). IRS-1 overexpression in HCC cell lines prevents transforming growth factor-β (TGF-β) induced
apoptosis (41). In fact, transfection of these cells with a dominant negative IRS-1 reverses
their malignant phenotype (42).
The role of IRS-1 in breast cancer has been difficult to elucidate, as both pro-host
and pro-tumor functions have been described. Simple over-expression of IRS-1 in MCF-7
breast cancer cells accelerates their growth, whereas IRS-1 gene silencing ultimately
results in apoptosis, at least under serum-free conditions (43, 44).
IRS-1 and IRS-2
transgenic mice both display enhanced tumor growth, metastasis, and resistance from
apoptosis (45). These mice develop mammary gland hyperplasia early in life, and display
unusual tumor histology, and not the typically encountered adenocarcinoma (46). Thus,
these over-expression studies may not be representative of pathophysiologic properties of
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IRS proteins in human cancers. As such, IRS-1 silenced tumor xenografts actually displayed
increased metastasis (47), consistent with a pro-host role for IRS-1. Additionally, studies of
IRS-1 in human breast cancer demonstrate that IRS-1 expression is lost in clinically
advanced cases (48).
We have recently described a pro-host role for IRS-1 in lung cancer (49). While
investigating the role of neutrophil elastase (NE) in lung cancer, we observed that NEdeficient tumors in the Lox-Stop-Lox-K-ras (LSL-K-ras) model of lung adenocarcinoma (50)
accumulated intracellular IRS-1 protein, whereas NE-sufficient tumors contained scant IRS1. We were able to demonstrate that IRS-1 is an intracellular proteolytic target for NE,
which induced cellular proliferation and pAkt production upon the degradation of IRS-1.
Ultimately, we discovered that the loss of IRS-1 functioned to increase the pool of bioavailable PI3K, rendering p85 free to interact with the more potent growth factors present
in lung cancer cells, especially the platelet-derived growth factor (PDGF) and receptor
(PDGFR) complex (51). Consistent with this concept, IRS-1 gene silencing in lung cancer
cells resulted in cellular proliferation and pAkt production whereas IRS-1 over-expression
induced cell cycle arrest. Furthermore, a correlation of the presence of NE with the
absence of IRS-1 was established in human lung adenocarcinomas. Thus, IRS-1 is capable
of both growth promoting and growth regulatory functions in cancers, depending on the
cell of origin.
Clinical-Translational Advances
With respect to IRS proteins, studies translating the findings observed in murine
models to human cancers are sparse.
However, the existing studies do support the
hypothesis that IRS-1 can function both for and against the host depending on the cell of
origin. Nearly all studies suggesting that IRS proteins function as positive effector of
growth factor were performed in metabolically active tissues, such as myocytes,
adipocytes, and hepatocytes (3). As such, IRS-1 appears to promote tumor growth in
malignancies that arise from such cell types including leiomyosarcomas, myosarcomas,
liposarcomas, rhabdomyosarcomas, and hepatocellular carcinomas (52). Interestingly, the
single study of IRS-1 in non-small cell lung cancer (NSCLC) demonstrated that the loss of
IRS-1 expression correlated with increased tumor growth (53), consistent with our
findings with respect to IRS-1 in murine models of lung adenocarcinoma. Further evidence
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that IRS-1 homeostatically regulates PI3K in cancer can be elucidated from the study of
G972R polymorphism. The presence of the G972R polymorphism within IRS-1 decreases
interaction with p85 (54), such that PI3K will be activated by other growth factors within
the cell. This polymorphism has been associated with an increased risk of prostate cancer
(55).
There are two potential clinical applications regarding IRS proteins in human
cancers: 1) use as a biomarker for PI3K +/- MEK/ERK activity and therapeutic response to
inhibitors of these respective pathways, and 2) indirectly maintaining IRS-1 levels within
tumor cells by inhibiting NE, which proteolytically degrades IRS-1 within lung cancer cells.
Inhibition of PI3K, of which IRS-1 is an integral component, is currently under
investigation in numerous early phase trials (56, 57). It is generally accepted that the
successful application of PI3K inhibitors (and all pathway directed therapies for that
matter) will require biomarkers predictive of tumor response to a given therapy. Early
studies employing targeted pathway inhibition in human cancers have already highlighted
this point. Specifically, the presence of K-ras mutation in colorectal cancer has been used to
predict response to anti-EGFR therapy (58) and Her2 over-expression has been used to
predict response to trastuzumab (herceptin) (59).
No biomarkers have been validated for response to PI3K inhibition. Some markers
are predictive of prognosis, such as combined analysis of PTEN and PIK3CA status, and may
yet prove useful in predicting response to PI3K antagonism (60-62). The first step towards
using IRS-1 and/or IRS-2 in this capacity will be to acquire the necessary translational
studies to clearly define the impact of the presence or absence of IRS-1/IRS-2 on PI3K
activity level, and to correlate these findings with meaningful clinical outcomes data for
each distinct cancer subtype. As described above, it is likely that IRS-1—PI3K interaction
will produce differential effects on cell behavior depending on the cell of origin. Therefore
it is plausible that IRS-1 levels will predict opposing outcomes when comparing HCC to
NSCLC, for example. We propose that the loss of IRS-1 in lung cancers will result in
paradoxical hyperactivity of PI3K independent of genetic mutations that commonly cause
PI3K hyperactivity (K-ras, PIK3CA, PTEN, etc.). PI3K hyperactive tumors (pAkt positive)
that are devoid of IRS-1 may represent a subclass amenable to inhibition of PI3K, and
possibly to MEK/ERK antagonism.
Commonly employed expression profiles will
continually fail to identify changes in IRS-1 expression, as IRS-1 is post-translationally
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degraded by components within the tumor microenvironment (NE). Therefore, detailed
analyses of IRS-1 protein content (IHC, Immunoblot) and gene expression (qPCR) must be
entertained to identify the nature of IRS-1 loss in each cancer type, and its impact on PI3K
activity and clinical outcomes.
The most logical way to manipulate tumor IRS-1 content for therapeutic benefit
would be to use an indirect approach. IRS-1 loss in human lung adenocarcinoma appears
to be as a result of neutrophil elastase-mediated degradation. Therefore, antagonism of NE
within the tumor microenvironment should preserve IRS-1 content within tumor cells and
maintain homeostatic regulation of PI3K, at least in lung tumors. We employed this
approach to successfully reduce lung tumor burden in LSL-K-ras tumor bearing mice using
the NE inhibitor, ONO-5046. NE inhibitors were previously developed as a potential
treatment for chronic obstructive pulmonary disease (COPD) and emphysema, however,
these agents were never fully tested given the difficult nature to perform clinical trials in
COPD (large cohort size, lack of adequate phenotypic markers to determine response, etc.)
and the likelihood that other elastin-degrading enzymes (i.e. matrix metalloproteinases)
would drive disease progression independent of NE (63-65). Despite these difficulties,
some NE inhibitors are currently undergoing early phase investigation in human COPD
(66), and if they prove safe, could readily be tested in human lung adenocarcinoma. Plans
to perform such studies are underway within our group.
Conclusions
IRS proteins interface with essential signaling pathways commonly implicated in
tumor development and progression. Current data suggest that IRS proteins may function
either for or against the host in the setting of cancer, depending upon the cell of origin.
Further translational studies will be required to determine the impact of IRS-1 and IRS-2
expression or loss on outcomes in various human malignancies. These data may prove
useful for the development of IRS protein levels as biomarkers of response to emerging
targeted pathway inhibiting therapies. Preservation of IRS-1 levels within lung tumor cells
by inhibiting the IRS-1 degrading enzyme, neutrophil elastase, may prove an effective
therapeutic strategy for patients with NSCLC.
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Figure 1. IRS-1 regulates downstream signaling of IR/ IGF-IR. IRS-1 is a homeostatic
regulator of PI3K signaling in lung tumor cells. IRS-1 has serine and tyrosine
phosphorylation sites that bind SH2 domain-containing proteins including p85, Grb2 and
SHIP2 among others. IRS-1 recruits PI3K and MEK/ERK via interaction with the regulatory
p85 subunit and GRB2, respectively. The catalytic subunit of PI3K, p110, is now available to
convert PIP2 to PIP3. PIP3 activates PDK-1, which subsequently phosphorylates AKT
enhancing cell survival, proliferation and growth. Downstream effectors of AKT inhibit
apoptosis via inhibition of Bad, Bim, Bax and caspase 9 and activation of BCL-XL and Bcl-2.
Tumor cell proliferation is promoted by the inhibition of GSK3, which targets cyclin D1 for
proteasomal degradation. Increased protein synthesis results from activation of the mTOR
pathway. The MEK/ERK pathway also promotes proliferation via interaction of IR/IGF-IR
with IRS or Shc proteins. A feedback loop exists between the PI3K/AKT signaling pathway
and IRS-1 in which IRS-1 is degraded through the ubiquitin-proteasome degradation
pathway. The above pathways have been well established in metabolically active tissues
including adipose and muscle. The above figure, however, describes an alternative to those
established paradigms. In lung cancer cells the PI3K/AKT pathway is weakly activated by
IRS-1. IRS-1 acts homeostatically to prevent activation of PI3K by more potent mitogens
including PDGF. Therefore, the loss of IRS-1 increases the amount of available PI3K, which
can then be activated by these mitogens causing much greater pathway activation. As
shown, neutrophil elastase (NE) can enter tumor cells and degrade IRS-1 during tumorassociated neutrophilic inflammation allowing other receptor tyrosine kinases (RTK) to
control PI3K signaling.
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RTK
IGF-IR/IR
NE
P85 IRS-1
PI3K
P110
PIP2
PTEN
Ras-GDP
Grb2
SOS
Ras-GTP
SHIP2
PIP3
SHIP2
Shc
Raf
PDK1
MEK
AKT
GSK-3
mTOR
S6
Bim
SK6
Bad
Protein Synthesis
Bax
Bcl-2
Caspase9
Cell Survival
ERK1/2
CyclinD
Bcl-xL
Growth and Proliferation
© 2011 American Association for Cancer Research
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Insulin Receptor Substrate Regulation of Phosphoinositol-3
Kinase
Heather E. Metz and A. McGarry Houghton
Clin Cancer Res Published OnlineFirst October 21, 2010.
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