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The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein
© Pearson et al.; licensee BioMed Central Ltd. 2012
Received: 26 March 2012
Accepted: 8 June 2012
Published: 8 June 2012
Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines.
NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined.
We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the ability of this oncoprotein to signal.
This is the first study demonstrating that the expression of immunophilin family co-chaperones is promoted by an oncogenic tyrosine kinase. Moreover, this is the first report establishing an important role for Cyp40 in lymphoma.
Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null cell immunophenotype [1–3]. This lymphoma primarily presents in children, adolescents, and young adults where it accounts for 10–20% of childhood non-Hodgkin lymphomas . ALK+ ALCL is characterized by the presence of chromosomal translocations involving the ALK gene, which encodes for a receptor tyrosine kinase belonging to the insulin receptor super-family. These translocations result in the expression of ALK fusion proteins that are critical for the pathogenesis of ALK+ ALCL [2, 3]. Moreover, ALK fusion proteins have been implicated in the pathogenesis of a subset of non-small cell lung carcinomas (ALK+ NSCLC) [4–7] and inflammatory myofibroblastic tumours (ALK+ IMT) [8–10]. In ALK+ ALCL several different ALK translocations have been described [2, 3]; however, the most common (~80%) is the t(2;5)(p23;q35) translocation involving the nucleophosmin (NPM) gene which generates the NPM-ALK oncogene [1–3].
NPM-ALK consists of the N-terminal region of NPM and the C-terminal kinase and intracellular domains of ALK [11, 12]. The NPM portion of this fusion protein possesses a dimerization domain required for the tyrosine kinase activity and transforming ability of NPM-ALK [13, 14]. The activity of the NPM-ALK oncoprotein is also critically dependent on the molecular chaperone, heat shock protein-90 (Hsp90) [15–18]. Hsp90 is a ubiquitously expressed protein that assists in the proper folding and activity of numerous cellular proteins [19, 20]. Hsp90 promotes the stability of NPM-ALK [15–18], as treatment of cell lines with the Hsp90 inhibitor, 17-Allylamino-Demethoxygeldanamycin (17-AAG), resulted in the proteasomal degradation of NPM-ALK . The treatment of ALK+ ALCL cell lines with 17-AAG resulted in cell cycle arrest and the induction of apoptosis [15, 18]; however, these effects are likely due to more than just decreased NPM-ALK levels. Hsp90 inhibition also decreased levels of the pro-survival serine/threonine kinase Akt, the cell cycle-associated proteins cyclin D1, cyclin-dependent kinase 4 (cdk4), and cdk6, as well as several other proteins in ALK+ ALCL [15, 18, 21]. The treatment of ALK+ ALCL cell lines with 17-AAG resulted in decreased phosphorylation of the serine/threonine kinase Erk without affecting Erk levels . Moreover, the treatment of ALK+ NSCLC with Hsp90 inhibitors resulted in Erk dephosphorylation as well as the degradation of Akt and the EML4-ALK oncoprotein in these tumours [22–24].
Hsp90 inhibitors are also effective at inhibiting EML4-ALK-driven tumourigenesis in vivo in the mouse [22, 23], and the treatment of three ALK+ NSCLC patients with the Hsp90 inhibitor, IPI-504, resulted in a partial response in two of the patients and stable disease in the other . Importantly, Hsp90 inhibitors are effective against tumour cells expressing ALK fusion proteins that possess mutations that render them resistant to the ALK inhibitor, Crizotinib [24, 26]. Thus, Hsp90 inhibitors may be useful in treating patients that develop resistance to ALK inhibitors.
One aspect of Hsp90 biology that is largely unstudied in ALK-expressing tumours is the role of Hsp90 co-chaperones. Many functions of Hsp90 are dependent on its association with co-chaperone proteins [19, 20]. Co-chaperones mediate various aspects of Hsp90 function, including the association of Hsp90 with client proteins and the regulation of Hsp90 ATPase activity [19, 20]. Cyclophilin 40 (Cyp40), FK506-binding protein (FKBP) 51, and FKBP52 are members of the immunophilin family of Hsp90 co-chaperones. This family is best characterized for its association with Hsp90-steroid hormone receptor complexes containing client proteins such as the glucocorticoid, estrogen, progesterone, and androgen receptors [27–30]. The individual immunophilin family members show some preference for specific hormone receptors, and they can both antagonize and promote the transcription mediated by these receptors. For example, FKBP51 inhibits the transcriptional activity of the glucocorticoid receptor [31–33], while FKBP52 is important for promoting the transcriptional activity of this receptor [32–35]. In addition to steroid hormone receptors, immunophilin co-chaperones have been found to complex with the Lck  and Fes  tyrosine kinases. As well, the expression and activity of ectopically expressed v-Src oncoprotein in Saccharomyces cerevisiae is dependent on the Cyp40 homolog, Cpr7 . Immunophilin co-chaperones are important in cancer, as Cyp40 and FKBP51 have been shown to promote the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines .
We identified Cyp40 in a mass spectrometry screen designed to identify proteins regulated by the JunB transcription factor in ALK+ ALCL (R.J.I and J.D.P; unpublished observation). JunB is an AP-1 family transcription factor that is highly expressed in ALK+ ALCL [40–42], and has been shown to promote the proliferation of the Karpas 299 ALK+ ALCL cell line . This transcription factor also promotes the expression of CD30 [44, 45] and the cytotoxic protein, Granzyme B , in ALK+ ALCL, which are phenotypic characteristics of this lymphoma [1, 47]. Since co-chaperone proteins are important for Hsp90 function, and Hsp90 activity is critical in ALK+ ALCL, we were intrigued by our observation that JunB might promote the expression of Cyp40 in ALK+ ALCL. In this study, we examined whether the expression of the immunophilin co-chaperones was regulated by oncogenic signalling in ALK+ ALCL. We also investigated whether the immunophilin co-chaperone proteins were important for the viability of ALK+ ALCL cell lines. We found that NPM-ALK induced the transcription of two immunophilin family co-chaperones, Cyp40 and FKBP52, but that only Cyp40 transcription was promoted by JunB. In addition, knocking-down the expression of Cyp40, but not FKBP51 or FKBP52, reduced the viability of ALK+ ALCL cell lines. However, knock-down of the immunophilin proteins did not appear to regulate NPM-ALK stability or activation. In conclusion, we demonstrate that some members of the immunophilin family of Hsp90 co-chaperone proteins are targets of NPM-ALK signalling, and that Cyp40 plays an important role(s) in ALK+ ALCL that is not shared by other immunophilin family co-chaperones.
Reagents and cDNA constructs
The monoclonal antibodies (mAbs) against JunB (C-11 and 204C4a), FKBP51, FKPB52, STAT3, phospho-STAT3 (Tyr 705), Myc, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). The Cyp40 polyclonal antibody was also from Santa Cruz Biotechnology. The anti-JunB (C-11) mAb was used for western blotting, while the anti-JunB (204C4a) mAb was used in EMSA experiments. The anti-tubulin mAb was from Calbiochem (San Diego, CA), the anti-ALK mAb from Dako (Burlington, ON, Canada), and the anti-phosphotyrosine mAb (4G10) was from Millipore (Billerica, MA). Anti-phospho-ALK (Tyr 338, 342, and 343 of NPM-ALK) and anti-Akt antibodies were purchased from Cell Signalling Technology (Danvers. MA). Short interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon RNAi Technologies (Lafayette, CO). The NPM-ALK inhibitor, Crizotinib, was generously provided by Pfizer [7, 48, 49]. To generate the human Cyp40 promoter–driven luciferase reporter construct, we PCR amplified the Cyp40 proximal promoter (−691 to +62 relative to the transcriptional start site) from the Karpas 299 cell line and cloned it into the pGL2 basic luciferase vector (Promega; Madison, WI). The AP-1 consensus sequence in the Cyp40 promoter was mutated from TGATTCA to TAACTAA to generate the AP-1 mutant construct. The Myc-tagged JunB construct was generated by adding a double myc tag to the 5′ end of the human JunB cDNA. This was then cloned into the pcDNA 3.1A eukaryotic expression vector (Invitrogen; Burlington, ON, Canada).
Cell lines and electroporations
The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol. For transfections involving siRNAs, 4 × 106 cells were transfected by electroporation with 100 nM pooled siRNA as previously described . Cells were then incubated for 48 h at 37°C prior to analysis. For luciferase reporter assays, 1 × 107 cells were transfected with 10 μg of the indicated pGL2 luciferase construct and 1 μg of a constitutively expressed Renilla luciferase construct (to control for transfection efficiency). In luciferase experiments involving siRNAs, cells were also transfected with 100 nM pooled control (non-targeting) or JunB siRNA. For luciferase assays performed on Karpas 299 cells over-expressing JunB, cells were transfected with the luciferase constructs as described above and 5 μg of Myc-tagged JunB or empty vector. Cells were then incubated for 24 h at 37°C prior to analysis of luciferase activity (see below).
Cell lysis, immunoprecipitations, and western blotting
Cells were lysed in Nonidet P-40 lysis buffer  containing protease inhibitor cocktail (Sigma-Aldrich; Mississauga, ON, Canada), 1 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate. Lysates were cleared of detergent-insoluble material by centrifugation at ~20,000 g for 10 min. The protein concentration of cleared lysates was determined using the BCA Protein Assay kit (Thermo Scientific; Waltham, MA). Anti-ALK immunoprecipitations were performed by incubating cleared lysates with 0.5 μg of the anti-ALK antibody and Protein A-Sepharose beads (Sigma-Aldrich) for 1–2 h at 4°C on a nutator. Beads were subsequently washed with lysis buffer and bound proteins eluted by boiling in SDS-PAGE sample buffer. Cell lysates or immunoprecipitates were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes. Western blots were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and band intensities quantified using a LI-COR Odyssey Infrared Imager (LI-COR Biosciences; Lincoln, NE). Expression of the quantified proteins were normalized to tubulin levels and expressed relative to control (non-targeting) siRNA-treated cells. The number of independent replicates for each experiment are indicated in the figure legends. To reprobe blots, membranes were stripped in 0.1% TBST, pH 2 prior to incubation with the new primary antibody.
Quantitative RT-PCR (qRT-PCR)
After collection using the RNeasy mini kit (Qiagen; Mississauga, ON), total RNA was digested with DNase I to remove potential DNA contamination, and then reverse transcribed to cDNA using the Superscript II Reverse Transcriptase System (Invitrogen; Burlington, ON, Canada). qRT-PCR was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences; Gaithersburg, MD) on an Eppendorf Mastercycler realplex4 thermal cycler. Cyp40 and FKBP52 mRNA levels were then determined using the ΔΔ-CT method  with β-actin as the housekeeping gene. The following primers were used: Cyp40 forward - TCGAGTCTTCTTTGACGTGGA, reverse - CAGTCGTGTGTCCAATGCCTT; FKBP52 forward - TGCTGAAGGTCATCAAGAGAGAG, reverse - ATGGTGGCTATGGCAATGTC; actin forward - AGAAAATCTGGCACCACACC, reverse - TAGCACAGCCTGGATAGCAA. Results are displayed relative to control siRNA-transfected cells and represent the mean and standard deviation of three independent experiments.
Luciferase assays were performed on a BMG Labtech Plate Reader using the Dual-Glo Luciferase Assay System (Promega) and the protocol provided by the manufacturer. Cyp40 promoter-driven firefly luciferase and constitutive Renilla luciferase activity were determined in triplicate for each sample. The level of firefly activity was normalized to Renilla activity and triplicate measurements were averaged. Three independent replicates were performed for each experiment.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were collected from Karpas 299 cells using the ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas; Burlington, ON, CA). EMSAs were performed with the LightShift chemiluminescent EMSA kit (Thermo Scientific) using a biotinylated probe corresponding to a 20 nucleotide sequence surrounding the AP-1 site of the Cyp40 promoter (TTGTACTGATTCATGTCTTT). The unlabeled AP-1 mutant competitor contained the same mutation as described for the luciferase reporter construct (see above). Binding reactions were performed with 7.5 μg of nuclear protein extract, 100 fmol of the Cyp40 promoter probe, and a 50-fold molar excess of an unlabeled Cyp40 promoter as a competitor. For super-shift experiments, 1 μg of the indicated antibody was pre-incubated with the reaction mixture for 15 min on ice prior to addition of the biotinylated probe.
MTS viability assays
After transfection with the indicated siRNAs, cells were resuspended to 4 × 104 cells/ml and incubated at 37°C for 48 h. The number of viable cells in each sample was determined in triplicate using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) (Promega). Triplicate measurements were then averaged and the percentage of viable cells determined relative to cells transfected with control siRNA. Each experiment was performed in quadruplicate.
Statistical analysis was performed using paired, one-tailed t-test in all cases, except the comparison of viability with Cyp40 siRNA to combined siRNA in which an unpaired, one-tailed t-test was performed.
JunB promotes Cyp40, but not FKBP51 or FKBP52, expression in ALK+ ALCL cell lines
NPM-ALK promotes Cyp40 and FKBP52, but not FKBP51, expression
Knock-down of Cyp40 reduces the viability of ALK+ ALCL cell lines
Cyp40 knock-down does not affect NPM-ALK levels or tyrosine phosphorylation, nor the tyrosine phosphorylation of cellular proteins in ALK+ ALCL
While this is the first report to show an important role for an immunophilin co-chaperone in lymphoma, several reports have demonstrated that this family of proteins perform critical functions in other malignancies. For example, knock-down of either Cyp40 or FKBP51 in prostate cancer cell lines decreased cellular proliferation; this was particularly evident in androgen-dependent cell lines where these co-chaperones promote the transcriptional activity of the androgen receptor . Metastatic melanoma has high levels of FKBP51, and knock-down of FKBP51 sensitized the SAN melanoma cell line to ionizing radiation . This response was postulated to be due to decreased anti-apoptotic signalling through NF-κB in response to reduced FKBP51 levels . In contrast, reducing the expression of FKBP51 in breast, lung, and pancreatic cancer cell lines resulted in reduced sensitivity to chemotherapeutic agents . It was suggested in this study that activation of Akt was partially responsible for this decreased sensitivity. Thus, the immunophilin co-chaperones perform important functions in a number of cancers, and may represent attractive therapeutic targets in some malignancies.
An important unanswered question arising from our study is why reducing Cyp40 expression in ALK+ ALCL cell lines resulted in reduced viability (Figure 5). Specific experiments to determine whether this is an increase in apoptosis, a decrease in proliferation, or combination of both of these processes have been inconclusive. This decrease in viability does not appear to be due to an impairment of NPM-ALK activity (Figure 6), and suggests that the dysregulation of another protein(s) is important for this phenotype. In addition to steroid hormone receptors and kinases, Cyp40 is known to associate with a number of other proteins with a variety of cellular functions including the c-Myb transcription factor , mutant forms of p53 , and the RACK1 scaffolding protein . Also, a genetic study in Arabidopsis identified an important role for the Cyp40 orthologue, SQUINT, in microRNA biogenesis . Thus, there are several cellular activities whose disruption could account for the decreased viability observed when Cyp40 is knocked down in ALK+ ALCL cell lines. Regardless of the exact cellular activity or activities regulated by Cyp40 that is important for the viability of ALK+ ALCL cell lines, our results clearly show these activities are not redundant with FKBP51 and FKBP52.
Our results show that Cyp40 does not regulate NPM-ALK levels or activity (Figure 6), but it is possible that other co-chaperones could be working with Hsp90 to regulate NPM-ALK activity. There are currently more than 20 known Hsp90 co-chaperones [19, 20]. One of these proteins, Cdc37, co-chaperones for many kinase client proteins including Erb-B2, c-Raf, CDK4, CDK6 and Akt . Cdc37 was identified by mass spectrometry as an NPM-ALK associated protein , and has also been shown to complex with EML4-ALK in NSCLC . These studies however, did not examine whether these interactions are important for the activity of the respective ALK fusion proteins. We are currently investigating whether Cdc37 or other Hsp90 co-chaperones influence NPM-ALK activity. If a co-chaperone protein that cooperates with Hsp90 to regulate NPM-ALK can be identified, it could represent a potential drug target to treat ALK+ ALCL, and other cancers expressing ALK fusion proteins, especially in situations where ALK mutations have resulted in resistance to conventional ALK inhibitors.
The Hsp90 chaperone protein regulates the NPM-ALK oncoprotein and other signalling molecules that promote proliferation and survival in ALK+ ALCL. Co-chaperone proteins are important co-factors of Hsp90, and in this study we examined the regulation and function of the immunophilin co-chaperones in ALK+ ALCL. We show that NPM-ALK is required for the expression of the immunophilin co-chaperones, Cyp40 and FKPB52, but not FKBP51 in ALK+ ALCL. Our findings further demonstrate that regulation of Cyp40 and FKPB52 by NPM-ALK is distinct, given that Cyp40 expression in ALK+ ALCL is promoted by the JunB transcription factor, whereas FKBP52 expression is not. Importantly, this is the first study demonstrating that signalling by an oncogenic tyrosine kinase promotes the expression of an immunophilin family co-chaperone, and identifies Cyp40 as a novel JunB transcriptional target. Finally, we demonstrate that Cyp40 promotes the viability of ALK+ ALCL cell lines in a manner that is independent of the other related immunophilin co-chaperones.
The authors would like to thank Jason Lee and Dr. Troy Baldwin for helpful discussions relating to this work, and Jason Lee and Brandon Maser for critically reading the manuscript.
This work was supported by operating grants from the British Columbia Proteomics Network, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Alberta Cancer Foundation/Alberta Innovates Health Solutions, and start-up funds from the University of Alberta (all to RJI). Part of this work was also funded by a grant from the University Hospital Foundation (to JTCB). JDP is a recipient of studentships from NSERC and the Alberta Cancer Foundation.
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