Src Homology 2 Domain Containing Inositol Polyphosphate 5-Phosphatases (SHIPs) as

Therapeutic Targets in Cancer, Immune Disease, and Metabolic Syndrome

Introduction______________________________________________________________________________________
Phosphoinositides (PtdIns) are a large family of phospholipids ubiquitous in
organismal cell membranes. The compounds consist of a glycerol backbone
linking two lipophilic polyacyl chains and a six carbon inositol sugar (Figure
1). The inositol ring exists in nine possible stereoisomeric forms, with the
myo- form the most common. Early studies on PtdIns indicated a central
role in various intracellular signalling processes in response to external
stimuli. The specific phosphoinositide PtdIns(4,5)P2, which is
phosphorylated at the 4 and 5 positions of the inositol ring, is a substrate
for phosphoinositide-specific phospholipase-C (PLC), which is activated by
the G-protein Gq though stimulation of G-protein coupled receptors such as
the 1-adrenergic and Histamine H1 receptors1. Activated PLC cleaves
PtdIns(4,5)P2 into inositol triphosphate (IP3) and diacylglycerol (DAG).
Inositol triphosphate is known to release intracellular Ca2+ through its
interaction with the IP3 receptor on the endoplasmic reticulum2. The
secondary metabolite DAG activates protein kinase C (PKC), which results in
further downstream signalling processes. Although the IP3/DAG pathway is
crucial to proper cellular function in most cells, PtdIns(4,5)P2 plays a role in
various signalling processes independent of the IP3/DAG pathway.
PtdIns(4,5)P2 can be phosphorylated by type I phosphoinositide-3-kinase
(PI3K) to produce phosphoinositol-(3,4,5)-triphosphate (PtdInsP3). Type I
PI3K acts upon PtdIns(4,5)P2 following stimulation via insulin or growth-
factor signals, and the product PtdInsP3 regulates a variety of metabolic,
proliferative, and apoptotic events through interaction with proteins
containing a pleckstrin homology (PH) domain3. The regulation of PtdInsP3
in cells is a target for modern pharmacological studies as PI3K and its
signalling molecules are upregulated in a variety of cancers cells4. Studies
into the PtdInsP3 signalling pathway revealed two enzymes responsible for
its degradation in vivo, the 3-phosphatase PTEN, and SH2 domain Figure 1: The generalized structure
containing inositiol polyphosphate 5-phosphatases (SHIPs). The two of phopshoinositide molecules,
enzymatic pathways are non-redundant, producing unique PtdIns products. containing: two polyacyl chains (A), a
PTEN is a 3-phosphatase, catalysing the production of PtdIns(4,5)P2 glycerol backbone (B), a bridging
whereas SHIPs cleave the 5-phopshate of PtdInsP3 to produce PtdIns(3,4)P2. phosphate (C), and myo-inositol (D).
The role of SHIPs in PtdInsP3 degradation and the nature of their enzymatic The carbons of the inositol ring are
products as independent signals have implicated the proteins as targets for labelled 1-6 for clarity.
therapeutic intervention in a variety of cancers as well as metabolic
diseases such as diabetes mellitus type II.

The Structure of Human SHIPs______________________________________________________________________

SHIP proteins are multidomain enzymes capable of hydrolysing the 5-phosphate of various polyphosphoinositide
species, particularly PtdIns(3,4,5)P3 and PtdIns(1,3,4,5)P4. Three conserved domains determine the main function of
the proteins. SHIPs possess an N-terminal Src Homology 2 domain, a central 5-phosphatase enzymatic domain, and a
C-terminal proline-rich domain. The Src Homology 2 domain binds strongly to phosphorylated tyrosine residues,
particularly those on receptor kinases and their protein intermediaries. Anderson et al.5 showed that fusion proteins
containing SH2 homology domains bind to autophosphorylated EGF and PDGF receptors following ligand stimulation.
The proline-rich domains possess AsnPro-Xaa-Tyr motifs which are phosphorylation sites and potential interaction
motifs for proteins possessing phosphotyrosine binding (PTB) domains6. Other motifs at the C-terminus such as Pro-
Xaa-Xaa-Pro are also implicated in protein interactions and downstream signalling; however, further experimentation
is needed to confirm these roles.
Current studies indicate two tissue-specific SHIPs exist in human cells, SHIP1 and SHIP2. Among these major families
exist several isoforms7, most likely produced through a variety of alternative transcriptional and splicing events. SHIP1
was originally isolated in haemopoietic cells and plays an important role in immune system regulation, cancer, and
stem cell function. The protein was characterized as a 145kDa tyrosine phosphorylated protein in complex with Shc (a
PTB domain protein), and Grb2 following stimulation of the B-cell receptor complex8.
A second enzyme, SHIP2, was isolated as a 155kDa protein containing the three principle SHIP domains in addition to a
sterile alpha motif (SAM) domain and a ubiquitin binding motif9. The SAM domain facilitates SAM-SAM interactions with
proteins such as Arf-GAP and Rho-GAP. The SHIP2 enzyme is widely expressed in human cells, with highest
concentrations in brain, skeletal, muscle and heart tissues.SHIP2 is thought to play an important role in regulation of
insulin signalling and obesity.

The Function of SHIP1 and Its Role in Cancer and Graft Versus Host Disease____________________________________
The enhanced resilience of cancer cells poses major problems for chemotherapeutic treatment of malignancies, and
recent studies have shown the role SHIPs play in signalling pathways leading to apopototic resistance.
Immunohistological staining studies on multiple myeloma bone marrow biopsies showed constitutive activation of the
PI3K/Akt signal pathway10, a pathway well characterized in survival and proliferative processes. The signalling pathway is
activated by ligand binding to a variety of receptors which may be G-protein coupled, tyrosine kinase dependant, or
cytokine dependant. Receptor stimulation recruits PI3K through interaction with the protein SH2 domain and
autophosphorylated tyrosine (pTyr) residues. These pTyr residues may be present on the receptors themselves or on
intermediary signalling proteins such as Gab1 and Jak1. Receptor mediated activation of PI3K increases the
concentration of intracellular PtdIns(3,4,5)P3, which recruits protein kinase B (Akt) through PH domain interactions.
Binding of Akt to the lipid induces conformational changes which exposure the main phosphorylation sites11. The
protein is then activated through the constitutive activity of phosphoinositide dependant kinase 1 (PDK1) (Figure 2).
Mayo and Donner et al.12 showed that Akt activation of the ubiquitin ligase Mdm2 causes relocalisation of the protein to
the nucleus where it targets the tumour suppressor p53 for proteasome degradation. In addition to p53 degradation,
Akt mediate phosphorylation of tumour suppressors p21 and p27 blocks nuclear relocalisation, resulting in increased
cyclin-dependant kinase (CDK) activity and lowered transcription of FasL, which increase cell proliferation and decrease
apoptotic signalling respectively13. These downstream processes associated with PI3K are likely to induce tumour
growth when deregulated.

Figure 2: The activity of SHIPs in relation to the PI3K/Akt signalling pathway. Stimulation of receptors such as cytokines or
receptor tyrosin kinases cause activation of PI3K, which increases intracellular PtdIns(3,4,5)P3 concentrations. The lipid is quickly
degraded by SHIPs to PtdIns(3,4)P2, which recruits Akt to the cell surface where it is phosphorylated by constitutively active PDK1.
Akt activates Mdm2 which stimulates p53 ubiquitination and degradation. Furthermore, Akt blocks nuclear translocation of the
cyclin and CDK inhibitors p21 and p27 as well as the forkhead transcription factor stalling transcription of fasL. The combination
of these downstream signals stimulates cell cycle activity and blocks apoptosis. The upregulation of this pathway is thought to
play a major role in cancer cell proliferation and their therapeutic resistance.
As the kinase activity of PI3K is counteracted by both PTEN and SHIP phosphatases; it was initially theorized that both
enzymes acted as tumour suppressors through their inhibition of the PI3K/Akt pathway. The tumour suppressing ability
of PTEN is well characterized, as knockout of gene pten in mice leads to myeloproliferative disorder resembling human
leukaemia14. Interestingly however, studies on the activity of SHIP1 did not show the same type of anti-tumour activity.
As PTEN and SHIPs have non-redundant enzymatic pathways, producing PtdIns(4,5)P2 and PtdIns(3,4)P2 respectively, the
products likely dictate divergent signalling processes. Franke
et al. showed through in vitro binding experiments with
various phosphoinositide species to Akt that the product of
SHIP hydrolysis, PtdIns(3,4)P2 bound Akt with a nearly 20
times higher affinity than PtdIns(3,4,5)P3 15. It is therefore
possible that SHIP increases recruitment and activation of Akt
and trigger tumorogenesis.
To test this hypothesis Brooks et al. carried out a series of
experiments on cells expressing SHIP1 to determine the
proteins role in apoptotic resistance16. High-throughput
screening of SHIP1 determined a small molecule, 3-
aminocholestane, as a potent inhibitor of enzymatic activity.
Acute myeloid leukaemia (AML) cell lines expressing SHIP1
were treated with the inhibitor to observe the effect on cell
survival. The AML cells showed a marked decrease in growth
and survival upon inhibitor treatment, whereas osteosarcoma
cells lacking innate SHIP1 activity showed no change in cell
viability (Figure 3). The decrease in cell viability was coupled
Figure 3: SHIP1 inhibition decreases the viability of
with decreased PI3K/Akt signalling and increased activation of
multiple myeloma cells in vitro. SHIP1 expression in
the apoptosis inducing protease Capsase 3 and ATP depleting
multiple myeloma (MG63) and osteosarcoma (KG1)
enzyme PARP. The addition of exogenous PtdIns(3,4)P2 to the
cells was analysed through western blotting (a).
inhibitor treated cells in subsequent experimentation showed
Both cell lines were then treated with varying
protective properties in a dose dependant fashion, implying
concentrations of 3- aminocholestane to
the product of SHIP1 activity as a key signal molecule in
determine the effect SHIP1 inhibition plays on cell
cancer cell survival. In addition to inducing apoptosis of
survival. The survival rate was followed by MTT
malignant cancer cells, the inhibition of SHIP1 in mice showed
assay (b).
changes in haemopoietic cell differentiation compared to
control groups. Daily administration of 3 -aminocholestane to SHIP+/+ mice resulted in increased myeloid
immunoregulatory (MIR) cell numbers in both the spleen and lymph nodes. The MIR cell numbers of SHIP1 inhibited
mice were similar to that of SHIP-/- mice, indicating analogous phenotypes. Prior experiments on SHIP-/- mice showed
increased MIR cell count alongside impaired priming of allogeneic T-cell responses17. Allogeneic T-Cell responses are
crucial pathways involved in to Graft vs. Host Disease and organ rejection. As mice treated with SHIP1 inhibitors showed
decreased T-cell response without the detrimental effects of full SHIP1 knockout, namely18, SHIP1 is a potential target
for immunosuppressant therapy following organ transplantation.

The Function of SHIP2 and Its Role in Metabolic Disease____________________________________________________

As SHIP2 is ubiquitously expressed in cells, it plays a more general role in cellular processes than the localized
homologue SHIP1. Batty et al. used Vanadate derived inhibitors of protein tyrosine phosphatases (PTPs) to examine the
effect on SHIP2 activity when a key regulatory enzyme is deactivated19. Vanadate-based inhibitors work analogously to
their physiological counterparts, reactive oxygen species, which are synthesized in response to a variety of cell signals
and inhibit PTPs through interaction with a redox active cysteine residue in PTP active sites. As PTP is known to
dephosphorylate SHIP2, inhibition of the protein both increased SHIP2 activity, and interestingly mediated SHIP2
localization to the plasma membrane through a currently unknown mechanism. This powerful activation mechanism,
stimulating a direct increase in SHIP2 activity alongside mediating relocalisation to the cell membrane, provides a
process by which cells can quickly switch the output of PI3K signalling from PtdIns(3,4,5)P3 to PtdIns(3,4)P2, amplifying
the downstream signalling processes of the PI3K/Akt pathway and leading to a breakdown of homeostasis.

Overactivity of SHIP2 mediated signalling has been shown to reduce the activation of insulin stimulated MAP Kinase and
Akt, which decreases the recruitment of GLUT4 receptor translocation to the cell surface. Deregulation of this pathway
points toward a potential mechanism for insulin resistance and development of diabetes mellitus type II20. Initial studies
on SHIP2-/- mice showed extreme insulin hypersensitivity, characterized by severe neonatal hypoglycaemia, deregulation
of gluconeogenesis, and perinatal death21. However, the deletion of SHIP2 encoding gene Inppl inadvertently deleted a
second gene Phox2a22. A more recent gene knockout specific for Inppl resulted in viable mice with normal glucose and
insulin levels, but which were also resistant to weight gain when put on a high fat diet23. Human genotype analysis has
since shown variations in the SHIP2 gene which may represent candidate mutations for predisposition to type II
diabetes. Studies carried out by Kagawa et al. aimed to determine nucleotide polymorphisms in the SHIP2 gene of a
diabetic cohort of the Japanese population24. A total of 10 polymorphisms were revealed, including 4 missense
mutations. A specific single nucleotide polymorphism SNP3 was located in the 5-phosphatase domain of the gene
sequence. The SNP3 variant was more common in control subjects than type II diabetics, stimulating further effort for in
vivo analysis of the enzyme activities. Transfection studies of the SNP3-SHIP2 variant showed that the protein inhibited
PI3K signalling activity less potently in expressing cells compared to the wild-type form, implicating a partial role of gene
polymorphisms in type II diabetes.

The Future of SHIP Targeted Therapy__________________________________________________________

As the role of SHIPs in a variety of disease states has just recently been elucidated, the targeting of such proteins in drug
therapy is poorly explored. The central role of SHIPs in a variety of crucial and signalling pathways poses a major
problem for the development of novel inhibitors, as unknown side-effects are likely to present upon treatment.
Furthermore, a crystal structure of SHIP1, possibly the most promising drug target, is yet to be solved, hindering a
rational drug design approach at the current time. Nevertheless, SHIPs currently provide an exciting avenue for further
biochemical and pharmacological research into novel therapeutics for the treatment of multiple diseases.

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