Wednesday, April 25, 2012

Pleckstrin Homology: A Spy Novel or a New Target for Melanoma

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 Phips 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 13 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 melanomapatients, 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.

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14.  Weinberg, R., Biology of Cancer, Garland (New York) 2008.