FOXO1 Is A Tumor Suppressor In Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) represents an unusual tumor entity where the tumor mass consists predominantly of stroma cells and infiltrating leucocytes. The malignant component, Hodgkin and Reed-Sternberg (HRS) cells comprise only about 1% of the tumor tissue. Classical Hodgkin lymphoma was defined as a subtype of B-cell lymphoma derived from germinal center or post-germinal center B-cells, which bear clonal Ig-gene rearrangements. Unlike in the majority of other B-cell lymphomas, the B-cell specific gene expression program is almost completely diminished in HRS. Several mechanisms including inactive B-cell specific transcription factors (E2A, PAX5, PU.1, OCT2, etc.), over-expression of transcription inhibitors (ABF-1, ID2, etc.), and epigenetic mechanisms (histone modifications and promoter hypermethylation) have been suggested to be responsible for this attenuation of the B cell-specific gene expression program. Another characteristic trait of cHL is the absence of common genetic aberrations responsible for malignant transformation. Mutations in tumor suppressors or oncogenes were rarely reported in cHL. Instead, cHL have acquired different alterations leading to constitutive activation of the pro-survival pathways, such as NF-κB, Jak-Stat, PI3K-AKT/PKB, ERK, API, and receptor tyrosine kinases (RTK) pathways. Critical downstream targets of the AKT/PKB pathway are the FOXO transcription factors; the latter can be phosphorylated and subsequently exported to the cytoplasm which eventually leads to the degradation of the FOXO proteins.Forkhead proteins are characterized by their highly conserved DNA-binding domain- the forkhead box. FOXO is one of the most studied subgroup of forkhead transcription factors. This family consists of four members FOXO1, FOXO3, FOXO4 and FOXO6. The activity of FOXO transcription factors is mainly regulated by covalent modifications. Several kinases have been identified that phosphorylate and inactivate FOXO proteins. These include AKT/PKB, the IκB kinase (IKK), SGK, and ERK. AKT/PKB phosphorylates three conserved amino acids in FOXO1, FOXO3, and FOXO4, which results in recognition by 14-3-3 proteins, nuclear export and in some cases degradation. In contrast, the AMPPK and JNK were suggested to induce nuclear translocation and activation of FOXO proteins. In addition, deacetylation by SIRT proteins was shown to critically regulate FOXO-activity.FOXO transcription factors regulate different cellular processes such as inhibition of proliferation, induction of differentiation or apoptosis and protection against oxidative damage. Among the specific FOXO target genes, cell cycle regulators like CCND2, CDKN1A and CDKN1B as well as pro-apoptotic genes like BIM, NOXA, and FASL were identified. On the other hand FOXO proteins may evoke pro-survival effects by stimulation of manganese superoxide dismutase or repressing TRB3, a pseudokinase, inhibiting AKT/PKB phosphorylation.For mature B cells (e.g., germinal center B cells), the survival signals provided by their antigen receptor (B cell receptor, BCR) are necessary to maintain viability and proliferation. BCR signaling activates pro-survival pathways such as NF-κB, PI3K-AKT/PKB, thereby protects B cells against apoptosis and promotes proliferation. Without a proper BCR signaling, B cells are doomed to go through apoptosis. Even with B cell lymphomas, BCR is also necessary for cell survival and proliferation. The only exception is Hodgkin lymphoma, tumor cells of which thrive without normal BCR signaling. Recent study shed light on this matter, researchers found out that while various downstream pathways including NF-κB cannot bypass the need of B cells for BCR, activation of PI3K or knockdown of FOXO1 can rescue BCR deficient B cells.Objective:Recent studies indicate that repression of FOXO1 plays a central role in BCR-dependent B cell survival and contributes to regulation of B-cell proliferation induced by other pathological and physiological stimuli. We set to investigate whether FOXO1 plays a role in B cell lymphomagenesis. We hypothesize that FOXO1 might play a tumor suppressing role in Hodgkin lymphoma.Methods:With help of Genesifter online software, we analyzed the expression profile of FOXO1 in various B cell subtypes and B cell lymphomas; the results were validated by qPCR, Western immunoblot and immunohistochemistry. Then the FOXO1 gene was ectopically expressed in cHL cell lines, the overexpression of the transgene was confirmed by Western immunoblot. The impact of FOXO1 expression on cHL cell line proliferation and survival was examined by FACS, etc. we examined the contribution of FOXO1 target genes to tumor suppressing functions of the FOXO1 protein using qPCR. Last but not the least; copy number changes of the FOXO1 locus on chromosome 13ql4.11 were investigated by Fluorescence in-situ hybridization (FISH) and Array Comparative Genomic Hybridization (aCGH)Results:In order to investigate a potential role of FOXO transcription factors, we surveyed the most recent and comprehensive microarray data of microdissected HRS cells of cHL samples. We compared expression levels of FOXO1, FOXO3 and FOXO4 in HRS cells and in normal B-lymphocytes sub-population mined from this database. Expression levels were determined with the help of Genesifter software. FOXO1 expression was higher than expression of FOXO3 and FOXO4 in B-cell subtypes, especially in centroblasts and centrocytes (CB+CC). Interestingly, in all cHL samples, FOXO1 levels were significantly lower than in normal B-cells subpopulations. The expression levels of FOXO3 and FOXO4 did not differ significantly between normal B-cells and HRS cells. To validate the microarray data on the FOXO1 down-regulation in HRS cells of cHL, we assessed FOXO1 and FOXO3 expression in tonsillar CD19+ cells, cHL cell lines, Burkitt's lymphoma (BL) cell lines and several other B cell lymphoma cell lines by Q-PCR. The results were in agreement with the gene expression array data. The relative expression of FOXO1 in CD19+ cells was much higher than the levels of FOXO3. Expression of FOXO1 was significantly higher in these primary non-malignant B-cells than in cHL cell lines. Interestingly, whereas the lowest levels of FOXO1 mRNA were detected in cHL cell lines, BL cell lines showed a somewhat intermediate expression level. We also assessed differences in FOXO1 protein expression between normal CD19+ B-cells and cHL cell lines by Western immunoblot. This analysis confirmed the Q-PCR data. FOXO1 protein expression in normal B-cells was significantly higher than in cHL cell lines.To rule out the possibility that low levels of FOXO1 expression in cHL cell lines were a consequence of in vitro propagation, we stained samples of hyperplastic tonsils, and cHL samples with FOXO1 antibodies. Tonsils demonstrated strong staining in the dark zone (DZ) of the germinal center containing a high density of centroblasts, as well as in the follicular mantle zone (MZ), where resting naive and memory B cells reside. Slightly weaker staining was seen in the germinal center light zone harboring centrocytes as the dominant cell population. Few FOXO1 positive cells were seen in the T-cell-zone (TZ). In 31 of 32 cHL cases analyzed, HRS cells were FOXO1 negative. Only one cHL sample revealed faint FOXO1 staining in HRS cells. In contrast, intense FoxO1 expression was found in a substantial fraction of the reactive bystander population surrounding the HRS cells. In nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) L&H cells were negative in 14 of 20 cases.To investigate the potential role of FOXO1 down-regulation in cHL pathogenesis, we used FOXO1 (A3)ER inducible constructs, A3 represents triple alanine mutations in serine and threonine residues. The substitutions make them resistant to AKT/PKB and SGK phosphorylation and inactivation, therefore the FOXO1 protein can be constitutively active. Furthermore the FOXO1 coding regions are fused in frame with the mutant ligand-binding domain of estrogen receptor allowing specific activation with 4-OHT. Stably infected cHL cell lines were derived and expression of FOXO1 (A3)ER was verified by Western immunoblot.We then investigated the influence of FOXO1 activation on growth of cHL cell lines. We found that activation of FOXO1 led to a significant reduction of live cells in all the cHL cell lines. FOXO proteins may inhibit proliferation and/or induce cell death, depending on cell types. In all cell lines, we saw reduced cell number in S-phase. The reduction of cells in S-phase was accompanied by an increase in G1/G0 fraction. We also analyzed apoptosis in the cells clones and found various levels of apoptosis induced by FOXO1.FOXO transcription factors have been shown to induce growth arrest by activating or suppressing specific target genes. We therefore determined expression levels of candidate FOXO target genes that could mediate these effects. CCND2 (cyclin D2) is required for cell cycle transition at the G1/S-transition and it is typically highly expressed in cHL. Activation of FOXO1 resulted in a two-fold down-regulation of CCND2 in cHL cell lines. In addition, expression of the cell cycle inhibitor CDKN1B (p27, Kip1) was increased in FOXO1 activated cells. In the attempt to identify causes of the FOXO1 repression, we found 13q14 deletions with involvement of the FOXO1 locus in 6 of 53 cHL cases (11.3%). In addition,4 of 5 cHL cell lines had losses of chromosome 13 or interstitial deletion of chromosome 13q.We found that inhibitor of AKT/PKB KP372-1 and inhibitor of MEK1/2 U01236 restored FOXO1 expression in cHL cells. The efficacy of the inhibitors correlated with the activation status of their specific targets. KP372-1 increased FOXO1 expression in exclusively in L1236 and in SUP-HD1 cHL cell lines, in which AKT was constitutively active. U0126 increased FOXO1 expression in KMH2, L428, UHO1, and SUP-HD1 cell lines demonstrating characteristic ERK activation, but not in L1236 cell line, in which ERK phosphorylation was much weaker. Our data suggest a critical role of FOXO1 repression in the pathogenesis of cHL.Conclusion:Here we show that FOXO1 is highly expressed in primary B-cells, but strongly down-regulated in HRS cells of cHL and in cHL-derived cell lines. This repression of FOXO1 transcription is associated with 13q14 deletions which involve the FOXO1 gene locus and could be critical for the cHL pathogenesis. Indeed, ectopic expression of PKB-independent alleles of FOXO1 resulted in growth inhibition and a certain level of apoptosis in cHL cell lines. As explanations of FOXOl proteins induced cell cycle arrest and apoptosis, the cell cycle inhibitors like p27 were induced, whereas cyclin D2 expression was repressed. In summary, our data indicate that repression of FOXO1 may represent an important event in cHL transformation.