There has been some recent work (see DeSemir et al) on the
targeting of the Pleckstrin Homology, “PH”, as an additional target for
controlling melanomas. As DeSemir et al state regarding the Pleckstrin Homology
Domain-Interacting Protein (PHIP) (slightly edited):
Given the important role of Akt in the IGF (Insulin
Growth Factor) axis, we then assessed whether Phip was involved in Akt
activation. …
Because of the uncharacterized role of PHIP in cancer, we
performed cDNA microarray analysis to identify the global patterns of gene
expression after suppression of Phip expression. Significance
analysis of microarrays identified 51 down-regulated genes (including
Igf2 and Tln1) and 184 overexpressed
genes … Thus, PHIP can regulate the expression of upstream mediators of the IGF
axis and downstream mediators of tumor cell invasion.
Having
demonstrated Phip’s functional role in promoting murine
melanoma metastasis, we examined its impact on human melanoma progression.
We performed immunohistochemical analysis of PHIP
expression on a tissue microarray cohort of 345 patients with primary cutaneous
melanoma …
High levels of PHIP expression were found in each
histological subtype of melanoma and accounted for almost one-third of the
melanomas in this cohort.
High PHIP expression correlated significantly
with the presence of ulceration, an adverse prognostic factor incorporated into
the staging classification for melanoma whose biologic basis is poorly
understood…
PHIP overexpression was significantly predictive of
reduced distant metastasis-free survival … and disease-specific
survival …
PHIP overexpression was an independent predictor of DMFS and DSS…
PHIP overexpression directly correlated with the
progression of distant metastases, and with reduced survival, in both murine
and human melanoma.
The human PHIP gene resides on the 6q14.1
locus. Deletions of the 6q arm have been shown in melanoma and have been suggested as a possible
diagnostic marker. …
FISH analysis revealed that the PHIP
locus was still present in all 78 melanomas examined.
Importantly, there was a significant correlation between PHIP
copy number (assessed as a percentage of cells with three or more
copies) and the corresponding PHIP immunohistochemical scores …
Melanomas with
immunohistochemical scores of 1–3 had a significantly higher
percentage of cells with increased copy number compared with melanomas with a
PHIP score of 0 .. In addition, 80.6% of PHIP 3 melanomas had three or more
copies of the PHIP locus.
Although we found no evidence of amplification,
because PHIP copy number remains comparable with chromosome
6 centromeric copy number increased copy number of the PHIP melanomas for β-catenin
mutations at six different sites (previously described in melanoma; COSMIC
database) and found no mutations at any of these sites.
These results show that PHIP levels can be activated in a
unique molecular subset of melanoma independent of mutations in these other
four genes.
This brief summary of the work makes PHIP an interesting and
attractive target. It presents a pathway element which is more a facilitator
rather than a major participant (see Weinberg). As we shall note later from
DeSemir et al, they contend that the PHIP target presents a more universal
target especially for those melanomas which do not have well defined mutations
in BRAF, NRAS or PTEN. As we have discussed previously, for example, PTEN
mutations, loss of control in the Akt pathway, is often an end game in cancer
progression, for example in prostate cancer and many others.
We will attempt to assemble some of the literature and
present a brief summary of this area. In many ways it is distinct from the
pathway targets themselves since the PH targets are smaller and often are found
in many of the pathway elements. The PHD. Pleckstrin Homology Domain, has
received significant interest by other researchers especially regarding its
pathway control effects. For example Hirano et al have examined it in CML and
Miyamoto et al in cardiology and the Akt pathway.
Pleckstrin and the Homology
We first examine Pleckstrin then its homology and its
function. We begin first with Pleckstrin. Pleckstrin is a specific protein which
is found in blood platelets. The name is derived using the concatenation of the
phrases: Platelet and LEukocyte C Kinase
substrate and the KSTR string of amino acids. It is located on 2p13.3.
Now the Pleckstrin Homology is defined as:
Pleckstrin homology domain (PH domain) is a protein
domain which consists of approximately 120 amino acids. The PH domain is
present in various proteins which are key elements of intracellular signaling as
well as constituents of the
cytoskeleton.
This domain can bind phosphatidylinositol lipids within
biological membranes (such as phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol
(4,5)-bisphosphate. PIP3 and PIP2), and proteins such as the βγ-subunits of
heterotrimeric G proteins, and protein kinase C.
Through these interactions, PH domains play a role in
recruiting proteins to different membranes, thus targeting them to appropriate cellular
compartments or enabling them to interact with other components of the signal
transduction pathways.
PH domains can be found in many different proteins, such as ARF.
Recruitment to the Golgi in this case is dependent on both PtdIns and ARF. A
large number of PH domains have poor affinity for phosphoinositides and are
hypothesized to function as protein binding domains. Proteins reported to
contain PH domains belong to the following families:
- · Pleckstrin, the protein where this domain was first detected, is the major substrate of protein kinase C in platelets. Pleckstrin is one of the rare proteins to contain two PH domains.
- · Ser/Thr protein kinases such as the Akt/Rac family, the beta-adrenergic receptor kinases, the mu isoform of PKC and the trypanosomal NrkA family.
- · Tyrosine protein kinases belonging to the Btk/Itk/Tec subfamily.
- · Insulin Receptor Substrate 1 (IRS-1).
- · Regulators of small G-proteins like guanine nucleotide releasing factor GNRP (Ras-GRF) (which contains 2 PH domains), guanine nucleotide exchange proteins like vav, dbl, SoS and S. cerevisiae CDC24, GTPase activating proteins like rasGAP and BEM2/IPL2, and the human break point cluster protein bcr.
- · Mammalian phosphatidylinositol-specific phospholipase C (PI-PLC) isoforms gamma
Discussion of PH in cancer is somewhat sparse and limited in
detail. Bunz has a short reference (p 191) and Weinberg also has passing
comments in several locations, and Schulz on p. 120.
PH and Pathways
The following is from Marks et al and shows how the PH
domain can act as a binding and activating substrate in the overall pathway
cascade process. It can unwrap from the complex protein of which it is a part,
and then it can attach to a membrane protein and this allows activation, in the
case below, by phosphorylating the resulting domain substrate. This simple
model offers also a mechanism to block pathway activation as well.
As Huang and Oliff state regarding the PH domain:
There are three members of the AKT (PKB) family. They are
widely expressed and implicated in apoptosis, insulin signalling and growth
regulation. All three contain a pleckstrin lipid-binding domain (PH Domain)and
are activated at the membrane by upstream kinases. Candidates for this upstream
regulatory activity include integrin-linked kinase, PDK-1, and possibly AKT
itself. In addition, AKT activity is regulated indirectly through modulation of
lipid metabolism.
The loss of PTEN (a protein and lipid phosphatase)
activity and the gain of PI3K (a protein and lipid kinase) activity correlate
with AKT activity and binding of AKT to the membrane lipid, PI(3)P. The PI3K
inhibitor wortmannin has already been shown to inhibit AKT signalling. Some
proteins that have been shown to be substrates of AKT and relevant to apoptosis
are listed. Antagonists of AKT kinase activity should inhibit signalling
through these downstream effectors.
We demonstrate this pathway selectivity and control below.
Here we have modified a Figure from Huang and Oliff to make the point that loss
of PTEN control or over-activation of the Akt pathway can result in excess of
proliferation and suppression of apoptosis. This is generalized below:
PTEN is a major control protein in pathway management. As
Chow and Baker had stated in an earlier description of the effects of PTEN:
Soon after the discovery of its PIP3 phosphatase
activity, PTEN was found to negatively regulate the PI3K/AKT pathway .
Generation of PIP3 by growth factor-stimulated PI3K activity results in
membrane recruitment of the serine–threonine kinase AKT via its pleckstrin
homology (PH) domain, and activation by phosphoinositide-dependent kinases
(PDK1 and 2) . Numerous AKT substrates have been identified affecting a broad
range of cellular activities .
A few that have been implicated in oncogenic
transformation include the Forkhead family of transcription factors (FOXO), p27KIP1, MDM2, GSK3, BAD, IKK-b, and tuberin
(TSC2), a negative regulator of mTOR. The specific targets phosphorylated by
AKT vary with physiological stimuli and cell context and the mechanism for this
selection is unclear. The complexity of this pathway is further underscored by
the recent finding that mTOR can act both upstream and downstream of AKT
activation. The raptor–mTOR complex can phosphorylate and activate AKT while the raptor–mTOR complex,
which regulates growth and protein translation, can be activated downstream of
AKT .
PTEN-mediated regulation of the PI3K/AKT pathway results
in cell context-dependent effects on cell size, proliferation and survival. A
dominant-negative form of AKT rescues the lethality caused by PTEN deficiency
in flies. This strongly suggests that
AKT is the major critical downstream target of PTEN activity ..
The impact of Akt has been understood now for quite a while
and the BRAF facilitation when mutated has become a focal element of the
control mechanism. However PH also plays a significant role and this too has
been understood. As Dehaia states:
PI3-kinase triggers signaling through multiple pathways,
many of which are thought to associate with cell growth and survival. PTEN,
working in opposition to PI3-kinase, is therefore associated with cell death or
arrest signals. Phospholipid residues such as PtdIns(3,4,5)P3 are present in cells upon stimulation by
several growth factors, such as platelet-derived growth factor (PDGF),
insulin-like growth factor (IGF), and epidermal growth factor (EGF).
Upon activation by growth factor, proteins
containing a pleckstrin-homology (PH) domain are recruited to the membrane 3
where they associate with phospholipids. One of the PH domain-containing
proteins relevant in this pathway is the serine-threonine kinase, AKT, also
known as PKB or RAC1. AKT, in turn, and as a consequence of
lipid binding, alters its conformation to allow two of its residues, threonine
308 and serine 473, to be phosphorylated and therefore become active.
The kinase responsible for phosphorylation of threonine
308 is phosphonositide-dependent kinase 1 (PDK1), an enzyme which also contains
a PH domain and is therefore dependent on lipid binding for its full activity.
There is some preliminary evidence, predominantly from in vitro studies, that a
second lipid-dependent, PH domain-containing enzyme, ILK (integrin-linked
kinase), is responsible for phosphorylation of the serine 473.
Further, a recent paper has proposed that the kinase
responsible for Ser 473 phosphorylation might in fact be PDK1, when it
associates with certain specific proteins, such as PDK1 interacting fragment
(PIF), as seen by in vitro studies. By dephosphorylating D3 residues on
PtdIns(3,4,5)P3 and PtdIns(3,4)P2, PTEN works in opposition to the PI3K/AKT
pathway and therefore counteracts cell survival mechanisms elicited by this
signaling. The mechanisms of cell survival associated with AKT appear to
involve multiple pathways, including growth factors, cytokines, c-myc
overexpression, UV irradiation, and matrix detachment.
One of the known signals activated by AKT is its
phosphorylation of the Bcl-2 family member, BAD: phosphorylation of BAD results
in suppression of apoptosis. AKT has also been reported to counteract the
apoptotic response of several cellular factors. Recently, the transcription
factor NF-kappaB has been implicated in the apoptotic response antagonized by the
PI3K/AKT pathway
Thus we have demonstrated that PH activateable proteins such
as Akt can be deactivated if it were possible to focus on the PH Domain as a
target sector. Recent work has demonstrated that in some detail.
Current Understanding
We now will examine some of the current understanding of PH
and its implications in melanoma specifically. We examine the work of two other
groups and then readdress that of DeSemir et al.
As Farang Fallah et al state:
As a major substrate of the insulin receptor, insulin
receptor substrate 1 (IRS-1) plays a central role in transducing insulin-dependent
signals that regulate biological processes such as cell growth and cellular
uptake of glucose. IRS-1 is a modular protein comprised of an N-terminal region
harboring a pleckstrin homology (PH) domain, followed by a phosphotyrosine
binding (PTB) domain that cooperatively ensures selective recognition and
efficient substrate phosphorylation by the activated insulin receptor (IR). The
C-terminal portion contains multiple tyrosine phosphorylation motifs which
serve as docking sites for the recruitment of various SH2 (Src-homology 2)
domain containing signaling molecules, such as phosphatidylinositol 3-kinase
(PI 3-kinase), Grb-2 adaptor protein, and SHP2 (SH2 containing phosphatase 2)
tyrosine phosphatase, which in turn elicit the activation of biochemical
cascades that promote the metabolic and growth responses to insulin….
In the present study we demonstrate that overexpression
of either PHIP or IRS-1 alone in muscle cells was not sufficient in promoting
transport of GLUT4 to plasma membrane surfaces This is consistent with other
observations, indicating that activation of IRS-1-associated signaling
effectors such as PI 3-kinase, although necessary, is not sufficient for GLUT4
activation.
Notably, growth factors such as platelet-derived growth
factor and interleukin-4 can activate PI 3-kinase as efficiently as insulin and
yet fail to stimulate glucose transport in insulinsensitive cells (17, 22).
One possible explanation is that additional PHIP/IRS-1/PI
3-kinase-independent pathways are required to coordinate GLUT4 intracellular
routing. Indeed, recent evidence points to a novel insulin-responsive pathway
that recruits flotillin/CAP/CBL complexes to IR-associated lipid rafts in the
plasma membrane, an event which is thought to potentiate GLUT4 docking to the
cell surface after IR activation.
Our data, however, provide support for the involvement of
PHIP/IRS-1 complexes in glucose transporter GLUT4 translocation in muscle
cells. Specifically, the use of DN-PHIP or IRS-1 PH domain constructs known to
interfere with efficient IR–IRS-1 protein interaction, and hence productive
signal transduction from IRS-1 to PI 3-kinase, blocked the ability of insulin
to stimulate GLUT4 mobilization in L6 myoblasts and inhibited
insulin-stimulated actin cytoskeletal reorganization, a process required for
the productive incorporation of GLUT4 vesicles at the cell surface. Moreover,
this inhibition did not coincide with changes in the autophosphorylation status
of the IR.
As Barnett et al state:
Akt/PKB (protein kinase B) is a serine/threonine kinase
which has a key role in the regulation of survival and proliferation [1–8].
There are three isoforms of human Akt (Akt1, Akt2 and Akt3) and they all have
an N-terminal PH (pleckstrin homology) domain and a kinase domain separated by
a 39-amino-acid hinge region. The PH domains have approx. 60%
identity and the kinase domains are >85% identical.
The hinge region is the least conserved at approx. 28%
identity. The Akt active-site residues, described in a recent report on the
crystal structure of Akt2 containing an ATP analogue and a peptide substrate ,
are the same in all three iso-enzymes. Based on the high degree of homology
between the AGC protein kinase family members, the identification of specific
active-site inhibitors has been predicted to be difficult. The identification
of Akt iso-enzyme-specific inhibitors seemed to be an even greater challenge….
Two Akt inhibitors were identified that exhibited
isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 while the second compound (Akt-I-1,2)
inhibited both Akt1 and Akt2 with IC50 values
of 2.7 and 21 μM
respectively. Neither compound inhibited Akt3 nor mutants lacking the PH
(pleckstrin homology) domain at concentrations up to 250 μM.
These compounds were reversible inhibitors, and exhibited
a linear mixed-type inhibition against ATP and peptide substrate. In addition
to inhibiting kinase activity of individual Akt isoforms, both inhibitors
blocked the phosphorylation and activation of the corresponding Akt isoforms by
PDK1 (phosphoinositide-dependent kinase 1).
A model is proposed in which these inhibitors bind to a
site formed only in the presence of the PH domain. Binding of the inhibitor is
postulated to promote the formation of an inactive conformation. In support of
this model, antibodies to the Akt PH domain or hinge region blocked the
inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be
cell-active and to block phosphorylation of Akt at Thr308 and Ser473,
reduce the levels of active Akt in cells, block the phosphorylation of known
Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis inducing
ligand)-induced apoptosis in LNCap prostate cancer cells.
We can now return to the results of DeSemir et al. As they
look to the usefulness of PHIP they state:
Although melanomas with mutant v-Raf murine sarcoma viral
oncogene homolog B1 (BRAF) can now be effectively targeted, there is no
molecular target for most melanomas expressing wildtype BRAF. Here, we show
that the activation of Pleckstrin homology domain-interacting protein (PHIP),
promotes melanoma metastasis, can be used to classify a subset of primary
melanomas, and is a prognostic biomarker for melanoma.
Systemic, plasmid based shRNA targeting of Phip inhibited
the metastatic progression of melanoma, whereas stable suppression of Phip in
melanoma cell lines suppressed metastatic potential and prolonged the survival
of tumor-bearing mice.
The human PHIP gene resides on 6q14.1, and
although 6q loss has been observed in melanoma, the PHIP
locus was preserved in melanoma cell lines and patient samples, and its
overexpression was an independent adverse predictor of survival in melanoma
patients. In addition, a high proportion of PHIP-overexpressing melanomas
harbored increased PHIP copy number.
PHIP-overexpressing melanomas include tumors with
wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and
phosphatase and tensin homolog, demonstrating PHIP activation in
triple-negative melanoma. These results describe previously unreported roles
for PHIP in predicting and promoting melanoma metastasis, and in the molecular
classification of melanoma.
This demonstrates the extended ability of PHIP to enhance
the usefulness of other markers. They continue as follows:
As a result, “triple-negative melanoma”
patients, whose tumors harbor wild-type v-Raf murine
sarcoma viral oncogene homolog B1 (BRAF), neuroblastoma
RAS viral (vras) oncogene homolog (NRAS), and phosphatase and
tensin homolog (PTEN) (the most common
mutations observed in melanoma), are not candidates for most targeted therapies
developed to date.
This as we have noted before is one of the most significant
findings. We know that BRAF mutations are currently targeted with some
beneficial albeit temporally limited results. Perhaps PHIP may add an
additional targeting.
They conclude:
Overexpression or mutation of genes that play important
roles in tumor progression. A high proportion of melanomas are characterized by
BRAF, NRAS, or PTEN mutations. However, the molecular basis of triple-negative
melanomas lacking these mutations is poorly characterized. Our results suggest
that PHIP levels may be used to classify some melanomas that lack these three
mutations. It is likely that additional molecular aberrations will be identified
to further characterize triple-negative melanomas.
Along with recent studies demonstrating that the IGF axis
is activated in melanomas with acquired resistance to BRAF inhibition (23),
these studies have identified IGF signaling as an
important alternative pathway to promote melanoma progression. Overall, our studies
identify PHIP as a molecular mediator of melanoma progression that also appears
to function in the setting of a subset of triple-negative melanomas.
Clearly BRAF, NRAS and PTEN mutations are well defined
targets, BRAF especially for melanoma and PTEN seems to span a wide number of
cancers. However if they are not changed the PHIP mutation seems more in line
with wit an reasonable target.
References
1. Barnett, S., et al, Identification and characterization of
pleckstrin-homology-domain dependent and isoenzyme-specific Akt inhibitors,
Biochem. J. (2005) 385, 399–408 (Printed in Great Britain) p 399.
2.
Biro, A., Class III
Phosphoinositide 3-kinase in Melanoma, Thesis, Univ Basel, 2011.
3.
Bunz, F., Principles of
Cancer Genetics, Springer (New York) 2008.
4.
Chow, L., S. Baker, PTEN
function in normal and neoplastic growth, Cancer Letters 241 (2006) 184–196.
5.
Dehaia, P, PTEN, a unique
tumor suppressor gene , Endocrine-Related Cancer (2000) 7 115–129,
6. DeSemir, D et al, Pleckstrin homology domain-interacting protein(PHIP) as a marker and mediator of melanoma metastasis, PNAS Early Edition, 2012.
7.
Farhang-Fallah,
J., et al, The Pleckstrin
Homology (PH) Domain-Interacting Protein Couples the Insulin Receptor substrate 1 PH Domain to Insulin Signaling
Pathways Leading to Mitogenesis and GLUT4 Translocation, MOLECULAR AND CELLULAR
BIOLOGY, Oct. 2002, p. 7325–7336.
8.
Hirano, I., et al,
Depletion of Pleckstrin Homology Domain Leucine rich Repeat Phosphatases 1 and
2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through
Continuous Phosphorylation of Akt Isoforms, Jrl Bio Chem V 284, 2009.
9. Huang,P., A. Oliff,
Signaling pathways in apoptosis as potential targets for cancer therapy, TRENDS
in Cell Biology Vol.11 No.8 August 2001.
10. Marks, F., Cellular Signal Processing, Garland (NY, NY) 2009.
11. McGarty, T., Prostate Cancer Genomics: A Systems Approach, DRAFT
http://www.telmarc.com/Documents/Books/Prostate%20Cancer%20Systems%20Approach.pdf
, 2012.
12. Miyamoto, S., et al, PHLPP-1 Negatively Regulates Akt Activity
and Survival in the Heart, Cir Res, 2010.
13. Schulz, W., Molecular Biology of Human Cancers, Springer (New
York) 2007.
14. Weinberg, R., Biology of Cancer, Garland (New York) 2008.
Posts
