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Annals of Clinical & Laboratory Science 35:131-136 (2005)
© 2005 Association of Clinical Scientists

Brief Communication

Morphoproteomic Analysis of Osteolytic Langerhans Cell Histiocytosis with Therapeutic Implications

Robert E. Brown
Division of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania

Address correspondence to Robert E. Brown, M.D., Division of Laboratory Medicine, Geisinger Medical Center, Danville, PA 17822-0131; tel 570 271 6332; fax 570 271 6105; e-mail rebrown{at}geisinger.edu.

Abstract

Morphoproteomics utilizes immunohistochemistry to identify protein analytes in tumor cells in order to uncover or confirm potential molecular pathways that may be essential to their proliferation, integrity, and histogenesis and that may serve as therapeutic targets. This communication illustrates the application of such an approach to osteolytic Langerhans cell histiocytosis and considers such molecular targets in the context of currently available therapies.

(received 19 January 2005; accepted 31 January 2005)

Keywords: Langerhans cell histiocytosis, morphoproteomics

During the past several years, we have been studying and attempting to characterize the protein circuitry in osteolytic Langerhans cell histiocytosis (LCH) in order to provide potential therapeutic options that are relatively nontoxic and appropriate to the disease process. Specifically, the demonstration of cyclooxygenase (COX)-2, farnesyl/geranylgeranyl transferase, and activated protein kinase C (PKC)-{alpha} have suggested and supported the use of indomethacin, aminobisphosphonates, and interferon (IFN)-{alpha}, respectively, in osteolytic LCH [1–5]. Recently, we have focused on defining signal transduction pathways that coincide with and integrate these previous observations, while exposing new therapeutic opportunities.

The immunohistochemical probes in this study included antibodies for the detection of the following proteins: platelet-derived growth factor receptor (PDGFR)-{alpha} (R&D Systems, Minneapolis, MN);PDGFR-ß (Santa Cruz Biotechnology, Santa Cruz, CA); p21ras (DAKO, Carpinteria, CA); phosphorylated (p)-extracellular signal-regulated kinase (ERK) 1/2 (phosphorylated at Thr 202/Tyr204), known as p44/42; p-Akt (phosphorylated at Ser 473); p-mammalian target of rapamycin (p-mTOR; phosphorylated at Ser 2448); p-p70S6K (phosphorylated at Thr 389); and p-nuclear factor-kappaB (p-NF-{kappa}B) p65 (phosphorylated at Ser 536). The anti-phosphorylated antibodies were all obtained from Cell Signaling Technology, Beverly, MA. With IRB approval, these antibodies were applied individually to sections from 3 representative cases of osteolytic LCH. This revealed cytoplasmic expression of both PDGFR {alpha} and ß, and of p21ras, predominantly cytoplasmic expression of p-Akt and p-mTOR, predominantly nuclear expressions of pERK 1/2 and p-p70S6K, and nuclear translocation of p-NF-{kappa}Bp65 (Fig. 1Go).



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  		Fig. 1. Osteolytic Langerhans cell histiocytosis (LCH) depicting osteoclasts and lesional LCH histiocytes with expressions of the following protein analytes in the latter: cytoplasmic immunoreactivities for PDGFR-{alpha} (upper left) and p21ras (upper right); predominantly nuclear immunopositivity for p-ERK 1/2 (middle left); primarily cytoplasmic localization of p-mTOR (middle right); nuclear and to some extent cytoplasmic compartmentalization of p-p70S6K (lower left); and nuclear translocation of p-NF-{kappa}Bp65 (lower right). Cytoplasmic expressions of PDGFR-ß and p-Akt (not illustrated) were comparable to PDGFR-{alpha}. (DAB chromogen; original magnifications x 600.)

 
Based on these data and previous observations by us and others, one can construct an integrated protein circuitry in LCH histiocytes that provides a basis for their growth and resistance to apoptosis and for osteoclastogenesis. Specifically, functional grouping of these protein analytes allows for the heterodimerization of PDGFR-{alpha} and PDGFR-ß [6] with signal transduction, as evidenced by downstream correlative expression of the phosphorylated serine /threonine protein kinases– p-Akt (Ser 473), p-mTOR (Ser2448), and p-p70S6K (Thr 389) [7–9]. Similarly, activation with translocation of PKC-{alpha} could be ascribed, at least in part, to PDGFR signaling through phospholipase C{gamma} [10–13] and in part through interleukin (IL)-1{alpha} [14] and aided by the prenylation and p21ras pathway [4,15,16] in LCH histiocytes. Growth promotion could be achieved through p-p70S6K-induced G1 cell cycle progression [17], following additional phosphorylation by p-ERK 1/2 [18]; and through activation (phosphorylation and nuclear translocation) of the transcription factor, NF-{kappa}B [19] consequent to the downstream signaling of PKC-{alpha}, p-Akt, immunophilins, and p-ERK 1/2. The latter signaling appears to be mediated in part through IkappaB kinase (IKK) activation [20–27]. Finally, there is the potential for cross-talk between the ras pathway and PI3-K/Akt pathway [28]. This protein circuitry is depicted in red in Fig 2Go.



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  		Fig. 2. Diagram of an osteoclast and the protein circuitry in an LCH histiocyte in osteolytic LCH, illustrating the findings in this study and incorporating relevant data from the literature (*). The molecular pathways created by the functional grouping of these proteins provide for a pathogenetic sequence (red color) leading to growth and prevention of apoptosis in the LCH histiocyte and to osteoclastogenesis. Specifically, this allows for: (1) the heterodimerization of PDGFR-{alpha} and PDGFR-ß with downstream signaling of PI3-K / p-Akt and parallel signaling through PKC-{alpha} and the p21 ras pathways; (2) cross-talk among the 3 pathways with convergent stimulation of PI3-K and its downstream effectors p-mTOR and p-p70S6K and the release of eIF4E leading to G1 cell cycle progression and translation of protein synthesis, respectively; (3) contributions by the ras / p-ERK 1/2 pathway to the activation (phosphorylation) of p-p70S6K and eIF4E; (4) activation and nuclear translocation of p-NF-{kappa}B consequent to the collaborative stimulation of IKK by PKC-{alpha} / p-Akt / immunophilins / p-ERK 1/2 and bcl-2; (5) anti-apoptotic protein synthesis effected by p-NF-{kappa}B and phosphorylated eIF4E; and (6) paracrine stimulation of osteoclastogenesis by IL-1{alpha} and IL-11 and by COX-2-generated prostaglandins. Opportunities for therapeutic intervention (blue color and (–) sign) include: Gleevec, pamidronate, IFN-{alpha}, rapamycin, Velcade / dexamethasone and indomethacin or combinations thereof.

 
Resistance to apoptosis is reflected in the expression of bcl-2 in LCH histiocytes [29,30]. Bcl-2 expression is upregulated by both p-NF-{kappa}B [31] and COX-2 [32], a downstream effector of PKC-{alpha} signaling [5]. Conversely, bcl-2 can mediate NF-{kappa}B activation via the ERK and IKK pathway [27]. Additionally, resistance to apoptosis in LCH histiocytes could be consequent to the phosphorylation, by p-mTOR and p-ERK 1/2, of 4EBP1 with release of eukaryotic initiation factor (eIF)4E and translation of antiapoptotic proteins [33–37]. Finally, osteoclastogenesis could be induced by multiple signals from the LCH histiocytes to include: IL-1{alpha} [5,38]; IL-11 [39]; and prostaglandins from COX-2 activity [2,40–42]. Once again, this circuitry is depicted in red in Fig 2Go.

Opportunities for therapeutic intervention and specific agents to interrupt the pathways include: STI571 (Gleevec; imatinib mesylate), an inhibitor of the PDGFR family of tyrosine kinases [43,44]; aminobisphosphonates to block farnesylation at the level of farnesyl diphosphate synthase and to promote osteoclastolysis [4,45,46]; IFN-{alpha} to take advantage of the PKC pathway, invoking an anti-LCH response [5]; rapamycin to interrupt immunophilin and p-mTOR signaling and thereby to retard growth and promote apoptosis [23,47,48]; Velcade (bortezomib), a proteosome inhibitor, in conjunction with dexamethasone to block the p-NF-{kappa}B and antiapoptic pathway [19,49,50]; and finally, indomethacin to inhibit COX-2 activity and reduce osteoclastogenesis [2,40]. These opportunities are depicted in blue in Fig 2Go. The reported efficacy of amniobisphosphonates [3,51], indomethacin [1], and IFN-{alpha} [5] in the treatment of LCH and most recently, imatinib mesylate (Gleevec [52]) in the treatment of cerebral LCH accords with this morphoproteomic analysis. The finding by Woltman and colleagues [53],that rapamycin induces apoptosis in monocyte- and CD34-derived CDla+ dendritic cells [53], is consistent with our finding of the activated mTOR pathway in LCH histiocytes. This pathway of convergence may be essential to the prevention of apoptosis in these lesional cells. Therefore, clinical trials that incorporate rapamycin may merit consideration in osteolytic LCH.

In summary, morphoproteomics applied to osteolytic LCH has enabled us to define the molecular circuitry in lesional histiocytes that could explain growth, resistance to apoptosis, and the promotion of osteoclastogenesis. In the process, it has exposed molecular pathways amenable to therapeutic intervention and has suggested several relatively nontoxic agents for the treatment of osteolytic LCH.

Acknowledgements

The author thanks Glen Kauwell and Laurie Kneller for technical assistance, and Sharon Coup for secretarial support and assistance with the graphics.

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