- Short report
- Open Access
HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells
© Schoof et al; licensee BioMed Central Ltd. 2009
- Received: 23 April 2009
- Accepted: 16 July 2009
- Published: 16 July 2009
In classical Hodgkin lymphoma (cHL) chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT) molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90) in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi) on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks). RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation.
- Tyrosine Phosphorylation
- L428 Cell
- HSP90 Inhibition
- Jak2 V617F
Recently, HSP90-inhibitors geldanamycin and its derivative 17-AAG have been shown to have anti-proliferative effects, sensitize for apoptotic stimuli and synergize with chemotherapy regimens in cHL cell lines [4, 5]. It was suggested that HSP90 inhibitors could be used for treatment of relapsed lymphoma patients in order to substantially reduce the toxic burden of standard therapies. HSP90 is a chaperone essential for maturation and stabilization of multiple client proteins. Thereby it is associated with crucial cellular functions like cell proliferation and survival by chaperoning key proteins of respective signaling pathways. The frequently observed overexpression of HSP90 in certain neoplasias probably leads to a stabilisation of proto-oncogenes or sustained aberrant signaling in the malignant cells. Jak1 and Jak2 have been described to interact with HSP90 and could therefore be involved in a permanent Jak-STAT signaling in cHL . Due to the overexpression of HSP90  and the permanent activation of Jak-STAT signaling [1–3, 7, 8], we asked the question if HSP90 supports permanent Jak-STAT signaling in cHL cells. Inhibition of HSP90 could be associated with the interruption of permanent STAT activation supporting the idea to use HSP90 inhibitors in multi-targeting tumor therapies. Therefore, we analysed the effects of the HSP90-inhibitor 17-AAG as well as RNA-interference mediated inhibition of HSP90 on the tyrosine phosphorylation of STAT1, STAT3, STAT5, STAT6 and protein expression of Jak1, Jak2, Jak3 and Tyk2 in cHL cells.
HSP90 has been associated with the stabilization of mutant forms of oncogenes including the Jak2 mutation V617F in hematologic diseases . Due to the observed permanent tyrosine phosphorylation of Jak2 and Tyk2 in cHL cell lines we analysed cHL cell lines for the presence of activating mutations. Jak2 V617F and Tyk2 V678F as well as Jak2 gene variations at D620E and E627E were sequenced within the cDNA of cHL cells as described previously [10–12]. In cHL cells lines L428, L1236, L591, KMH2, HDLM2 and L540 Jak2 is transcribed from the allele characterised by V617, D620 and E627, therefore no V617F activating mutations or other changes could be observed in the transcripts. The absence of Jak2 activating mutation V617F has recently been published . The homologous mutation of Jak V617F in Tyk2 named V678F has been found to activate Tyk2 in the same manner as Jak2 V617F . In all analysed cHL cell lines (L428, L591, L1236 and HDLM2) no Tyk2 V678F mutation was detected. Moreover, no other mutations within the sequenced cDNA regions were detected, suggesting no stabilization of mutant forms of Jak2 and Tyk2 by HSP90. This observation is in line with recent reports of autocrine activation of Jak-STAT signaling in cHL cells [1, 14].
In summary, this study shows that HSP90 is important for the permanent activation of STAT molecules in cHL cells. Moreover, we conclude that the reduced protein levels of the Jaks upon inhibition of HSP90 are a major reason for the decreased STAT phosphorylation after inhibition of HSP90 by 17-AAG or RNAi against HSP90. Therefore, not only Jak1 or Jak2 as proposed by Shang and colleagues but also Jak3 and Tyk2 might be stabilised by HSP90 . As proposed by Cochet and co-workers the activation of Jaks correlates with STAT activation in cHL cells . This is in line with our finding that reduced protein amounts of Jaks lead to a reduced tyrosine phosphorylation of STATs in cHL cells. Nevertheless, we can not rule out that other HSP90 client proteins affect the phosphorylation of the STAT molecules or the expression of the Janus kinases, but HSP90 inhibition is accompanied by a strong reduction of permanent Jak-STAT signaling shown to be essential for cHL cell proliferation. Therefore, it is likely that the reduced cell proliferation upon HSP90 inhibition is mainly mediated by abrogation of permanent STAT activation.
Constitutive activation of NF-κB is another hallmark of cHL and could promote the expression of IL-13 and therefore activation of STATs . The aberrant HSP90 expression in cHL cells could also be mediated by NF-κB and represent an additional mechanism to support permanent activation of STATs . Thereby NF-kB could contribute to the malignant phenotype of cHL cells due to the stabilization of key proteins of multiple signaling pathways by HSP90 upregulation. Georgakis and co-workers showed an inhibition of PI3K/Akt and MEK pathways by 17-AAG in cHL cells which have been shown to contribute to survival of cHL cells to some extend . The data presented here in conjunction with the recent publications outlines HSP90 as a potent therapeutical target in cHL patients of advanced stages or relapse. The abrogation of STAT phosphorylation of by HSP90 inhibition may be also useful for other cancer entities with permanently activated STAT molecules. Orally administered HSP90 inhibitors may open new perspectives in the treatment of these lymphoma by targeting multiple pathways and therefore eliminating the danger of resistance development observed in single targeted therapies as with imatinib mesylat.
We would like to thank Dr. Martina Vockerodt and Alexandra Schrader for helpful discussions and critical review of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through the Graduiertenkolleg 1034 http://www.gcpg.de.
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