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Phosphatase and Tensin Homolog on Chromosome 10 Is Phosphorylated in Primary Effusion Lymphoma and Kaposi’S Sarcoma

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Phosphatase and Tensin Homolog on Chromosome 10 Is Phosphorylated in Primary Effusion Lymphoma and Kaposi’S Sarcoma The American Journal of Pathology, Vol. 179, No. 4, October 2011 Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.ajpath.2011.06.017 Tumorigenesis and Neoplastic Progression Phosphatase and Tensin Homolog on Chromosome 10 Is Phosphorylated in Primary Effusion Lymphoma and Kaposi’s Sarcoma Debasmita Roy* and Dirk P. Dittmer*† ated with Kaposi’s sarcoma-associated herpesvirus 2–4 From the Curriculum in Genetics and Molecular Biology,* and the (KSHV) infection. KSHV, known formally as human Department of Microbiology and Immunology,† Lineberger herpesvirus 8 (HHV-8), shows primary tropism for B Comprehensive Cancer Center, Center for AIDS Research, cells and endothelial cells. In addition to KSHV infec- University of North Carolina at Chapel Hill, Chapel Hill, tion, PEL cells can be coinfected with Epstein-Barr North Carolina virus [EBV; formally known as human herpesvirus 4 (HHV-4)].5 KSHV is also the causative agent of Kaposi’s sar- coma (KS), a tumor of endothelial cell lineage. KS is Primary effusion lymphoma (PEL) is a non-Hodgkin’s characteristically seen in HIV-infected patients, but B-cell lymphoma driven by Kaposi’s sarcoma-associ- also occurs with reduced frequency in other settings of ated herpesvirus. It is uniquely sensitive to mTOR, immune suppression (eg, in solid organ transplant re- PI3K, and Akt inhibitors; however, the basis of this cipients).6 In addition, KS is found in elderly patients in requirement for the mTOR pathway remains to be the absence of overt immune deficiency (classic KS) elucidated. The phosphatase and tensin homolog and in children in KSHV-endemic regions, such as gene (PTEN) on chromosome 10 controls the first Sub-Saharan Africa. Stallone et al7 reported on a series step in the phosphatidylinositol 3 kinase (PI3K)-Akt- of transplant patients who developed KS. On switching mammalian target of rapamycin (mTOR) pathway from cyclosporine to another immunosuppressant, and is genetically inactivated in many solid tumors. rapamycin, the KS lesions regressed; however, the We find an absence of PTEN deletions, mutations, same patients maintained normal graft function, be- or protein mislocalization in PEL. However, we find cause rapamycin maintained T cell-targeted immuno- consistent hyperphosphorylation at serine 380 of 7 PTEN, which is an inactivating modification, in PEL suppression . Multiple studies have confirmed this 8–11 cell lines and in tumor xenografts. We also evalu- finding, although others have described cases of ated a large tissue microarray of Kaposi’s sarcoma KS and PEL that responded only partially or did not 12–16 biopsies and observed concordant high levels of respond to rapamycin. There has also been spec- phospho-PTEN, phospho-Akt, and phospho-S6 ribo- ulation on whether rapamycin acts in a direct or indi- 17 somal protein. Reintroduction of PTEN into PEL in- rect manner via immune modulation only. Thus, there hibited colony formation in soft agar, verifying the is a need to understand the molecular mechanism of functional dependence of PEL on PI3K signaling. both rapamycin and its analogs in PEL and KS, and to This was also true for PEL cell lines that carried evaluate potential biomarkers that could help in pre- mutant p53 and for KS-like cell lines. Activating dicting response. PTEN in these cancers may yield a new treatment strategy for PEL, KS, and similar PTEN wild- Supported by NIH grants DE018304 and CA163217 (D.P.D.). TMA con- type lymphomas. (Am J Pathol 2011, 179:2108–2119; struction was funded by NIH grant NCI CA066529. DOI: 10.1016/j.ajpath.2011.06.017) Accepted for publication June 28, 2011. Supplemental material for this article can be found at http://ajp. amjpathol.org or at doi: 10.1016/j.ajpath.2011.06.017. Primary effusion lymphoma (PEL) was initially identified Address reprint requests to Dirk Dittmer, Ph.D., Department of Micro- as an AIDS-associated non-Hodgkin’s lymphoma with biology and Immunology, Lineberger Comprehensive Cancer Center, 1 a poor prognosis. PEL also occurs in the absence of Center for AIDS Research, University of North Carolina at Chapel Hill, CB# HIV infection (eg, in transplant recipients taking im- 7290, 715 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7290. E-mail: mune-suppressive drugs).2,3 PEL is strongly associ- [email protected]. 2108 PTEN in PEL and KS 2109 AJP October 2011, Vol. 179, No. 4 Table 1. Summary of PEL Cell Lines, with Literature Citations Lymphoma Cell line type KSHV EBV p53 PTEN Rapamycin sensitivity BCP139 PEL ϩϪS262; homozygous insertion Wild type Ͻ50 nmol/L BC140 PEL ϩϩWild type Wild type Ͻ10 nmol/L BC240 PEL ϩϩWild type* Wild type Ͻ50 nmol/L BC341 PEL ϩϪWild type Wild type Ͻ50 nmol/L BC542 PEL ϩϩWild type* Wild type Ͻ50 nmol/L BCBL143 PEL ϩϪM246I; heterozygous mutation Wild type 5.5 mmol/L BCLM44 PEL ϩϪWild type Wild type Ͻ50 nmol/L BJAB45 Burkitt’s ϪϪDeletion; nonfunctional Wild type Ͻ1 nmol/L DG7546 Burkitt-like ϪϪR283H; heterozygous47 Wild type Not tested JSC148 PEL ϩϩWild type Wild type Ͻ50 nmol/L TY149 PEL ϩϪS262; insertion; M246I Wild type Ͻ50 nmol/L VG150 PEL ϩϪWild type Wild type Ͻ50 nmol/L *Present study. Rapamycin binds to and inhibits the activity of mam- Materials and Methods malian target of rapamycin complex-1 (mTORC1),18 a downstream effector of the phosphatidylinositol-3-kinase Cell Culture (PI3K) signaling cascade. Phosphorylation of receptor B-cell lines were cultured in RPMI Medium 1640 supple- tyrosine kinases results in activation of the p110 catalytic mented with 100 ␮g/mL streptomycin sulfate, 100 U/mL subunit of phosphatidylinositol 3 kinase (PI3K), which penicillin G (Invitrogen Life Technologies, Carlsbad, CA), then phosphorylates membrane-associated phospho- 2 mmol/L L-glutamine, 0.05 mmol/L 2-mercaptoethanol, lipid phosphatidylinositol-4,5-biphosphate (PIP2)tobe- 0.075% sodium bicarbonate, 1 U/mL IL-6 (PeproTech, come phosphatidylinositol-3,4,5-triphosphate (PIP3). Rocky Hill, NJ), and 10% fetal bovine serum (Mediatech, PIP3 recruits the protein serine-threonine kinase Akt to the Manassas, VA) at 37°C in 5% CO2. The cell lines are plasma membrane. Akt is phosphorylated on serine 473 characterized in Table 1. by mTORC2 and by PI3K-dependent kinase 1 (PDK1) on threonine 308. Activated Akt then phosphorylates down- Comparative Genomic Hybridization Analysis stream targets, mTORC1 being one of its effectors.19–23 The protein encoded by the phosphatase and tensin Genomic DNA was isolated from the PEL cell lines using homolog gene (PTEN), located on band q23 of chromo- a Wizard genomic DNA purification kit (Promega, Madi- some 10, constitutes a key negative regulator of the PI3K- son, WI) according to the manufacturer’s protocol. mTOR signaling pathway. It can revert phospholipid PIP3 Genomic profiles were obtained by hybridizing the DNA to PIP2, thereby counteracting the activation of Akt by to GeneChip 250K Nsp and 250K Sty human mapping PDK1.19,22,24–30 PTEN is frequently mutated in human arrays (Affymetrix, Santa Clara, CA), which contain mark- cancers.22,24–28,30 Ectopic expression of PTEN in tumor ers for copy number determination, in addition to single cells results in cell cycle arrest or apoptosis.31,32 PTEN nucleotide polymorphisms. All data were analyzed using exists either in a hypo- or hyperphosphorylated state, the Partek Genomics Suite version 6.0 (Partek, St. Louis, corresponding to active (open) and inactive (closed) en- MO). Raw data were imported and adjusted for back- zymatic states, respectively.33 PTEN expression and ground using a robust multiarray algorithm. Copy num- phosphorylation status thus serve as markers for PTEN bers were assessed using the software suite’s unpaired activity. Various solid tumors that require PI3K-mTOR sig- analysis and were compared with baseline created using normal tonsil control DNA. Copy number variation was naling have either a deletion or mutation in PTEN34; how- represented in both the heat map and the dot-plot profile ever, the status of PTEN in PEL and KS remains a subject along the length of the individual chromosomes using a of investigation. Our research group has previously re- visualization scheme from the Partek software suite. ported that rapamycin inhibits PEL in culture, in a mouse model, and in a patient.35 These findings suggest that PEL belongs to a class of cancers, such as Mantle Cell PCR and Sequencing Lymphoma, that depend on PI3K-Akt-mTOR signaling. Genomic DNA was isolated using a Wizard genomic KSHV proteins may activate mTORC1 through cellular DNA purification kit (Promega). PCR amplification was receptor engagement (by vIL6) or through PI3K activa- performed using GoTaq PCR master mix (Promega). 36–38 tion (by viral proteins such as vGPCR and K1), fur- Specific primers, flanking individual exons, were as ther corroborating the dependence of KSHV-infected given in Table 2. The amplified fragments were sub- cells on PI3K-mTOR signaling. In the present study, we jected to Sanger sequencing. mRNA expression was examined whether, in addition to viral factors, host ge- determined using cDNA subject to RT-PCR, using netic aberrations in PTEN contribute to the required ac- primers as given in Table 2. cDNA was synthesized tivity of the PI3K-Akt-mTOR pathway in PEL and KS. from total RNA isolated from PEL cells using TRIzol 2110 Roy and Dittmer AJP October 2011, Vol. 179, No. 4 Table 2. Primer Sequence and Annealing Temperature for PCR Analysis, with Literature Citations Annealing temperature, Exon Forward primer Reverse primer
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