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Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2

Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2 www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 22 Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2 1 2 1 1 1 Aine McCarthy , Jacek Marzec , Andrew Clear , Robert D. Petty , Rita Coutinho , 1 1 1 1 Janet Matthews , Andrew Wilson , Sameena Iqbal , Maria Calaminici , John G. 1 1 Gribben and Li Jia Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Centre for Molecular Oncology2, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Correspondence to: Li Jia, email: L.jia@qmul.ac.uk Keywords: Autophagy, BCL-2, follicular lymphoma, autophagy PCR array, and tissue microarray Received: October 09, 2014 Accepted: October 18, 2014 Published: October 18, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL- + - 2 /BCL-2 lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the + - BCL-2 Su-DHL4 cell line compared with BCL-2 Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression proles fi were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease. and inhibition of autophagy can enhance tumor cell death INTRODUCTION by diverse anticancer therapies [9-11]. Follicular lymphoma (FL) is the second most Macroautophagy (hereafter referred to as common lymphoma diagnosed in the United States and autophagy) is a physical and pathological process that Western Europe, accounting for approximately 20% of all allows eukaryotic cells sequester portions of cytoplasm non-Hodgkin lymphomas (NHLs) and 70% of indolent to form autophagosomes and target them for degradation lymphomas and is generally considered incurable [12, through the fusion of autophagosomes with lysosomes 13]. The t(14;18)(q32;q21) translocation characterizes where they are degraded and recycled [1-4]. Growing approximately 85% of FL and 20% of diffuse large evidence demonstrates that autophagy plays important and B-cell lymphoma (DLBCL) and results in constitutive paradoxical roles in tumorigenesis and in the treatment of overexpression of the anti-apoptotic protein BCL-2 [14, cancer [4-7]. Autophagy can suppress tumorigenesis by 15]. Overexpression of BCL-2 in NHLs plays important removing damaged organelles/proteins and limiting cell roles in disease pathogenesis and resistance to apoptosis. growth and genomic instability [6]. In contrast, induction It is currently unknown whether BCL-2 plays an important of autophagy by metabolic stress in apoptosis deficient role in regulation of autophagy in indolent FL and more tumor cells can support tumor cell survival [8]. Abundant aggressive DLBCL. preclinical evidence indicates that stress-induced The role of the anti-apoptotic protein BCL-2 in autophagy in tumor cells is predominantly cytoprotective www.impactjournals.com/oncotarget 11653 Oncotarget autophagy is currently debated and remains unclear. DHL4 cell line was also distinguishable from Su-DHL8 BCL-2 has been proposed as a major binding partner of cells based on its autophagy-related gene expression Beclin-1 which it binds in a nutrient-dependent manner, profile (GEP) (Figure 1 B and Suppl Figure 1). Four consequently down-regulating levels of starvation- autophagy machinery and nine autophagy regulatory genes mediated autophagy [16]. Recently, it was reported that were up-regulated in the Su-DHL4 cell line compared to the anti-apoptotic BCL-2 family members do not directly Su-DHL8 cells (Figure 1 C and D). These data support inhibit components of the autophagic pathway but instead that BCL-2 Su-DHL4 cells may have higher basal level affect autophagy indirectly, owing to their inhibition of autophagy activity compared with BCL-2 Su-DHL8 cells. Bax and Bak [17]. Autophagy is often activated as an We next sought to determine the capacity of + - adaptive response against ER stress [18, 19]. In cancer the autophagic flux in both BCL-2 and BCL-2 cells. cells, metabolic stress strongly induces autophagy which is Inhibition of the autophagic flux by treatment of cells sustained when apoptosis is blocked [8, 20, 21]. Questions with CQ [7, 25] or induction of autophagy by nutrient raised over the roles of BCL-2 in autophagy are: whether deprivation led to increased or decreased p62 and LC3B-I/ overexpression of BCL-2 in human tumors inhibits both LC3B-II protein expression respectively in both cell apoptosis and autophagy; and whether inhibition of lines (Figure 2 A and B), demonstrating that autophagy apoptosis by overexpression of BCL-2 could activate activity was not suppressed in the BCL-2 Su-DHL4 cells. the autophagic pathway in favor of prolonged tumor cell Beclin-1 expression did not change in response to either survival. treatment, indicating that its levels are not governed by The role of BCL-2 in autophagy in human NHLs the autophagy flux. Both Su-DHL4 (Figure 2 C and E has not previously been reported. We hypothesize that and Suppl Figure 2) and Su-DHL8 (Figure 2 D and F and overexpression of BCL-2 in FL may cause an increased Suppl Figure 3) cell lines responded to nutrient deprivation autophagy activity due to suppression of apoptosis. In this by up-regulating and down-regulating autophagy-related article, we aimed to determine whether overexpression genes. Among them, SQSTM1 (p62) and CDKN1B genes of BCL-2 could alter autophagy status in BCL-2 positive showed significantly increased expression in both cell + - (BCL-2 ) and negative (BCL-2 ) DLBCL cell lines, lines (Figure 2 E and F). Between 2 and 6 hours starvation, primary FL, DLBCL and reactive (RA) samples using we also noted increased p62 protein expression despite both autophagy RT Profiler PCR Array and tissue increased autophagic degradation (Figure 2 B). A similar microarray (TMA). We demonstrate for the first time that phenomenon has also been observed in mouse embryonic overexpression of BCL-2 does not inhibit autophagy in fibroblasts [26]. Increased expression of CDKN1B in human FL. response to starvation may cause cell cycle arrest in the G1 phase [27]. In addition, the Su-DHL4 cell line also showed significantly increased expression of key RESULTS autophagy machinery genes including GABARAPL1, GABARAPL2, MAP1LC3B (LC3B) and CTSS (Figure 2 C + and E). These results suggest that BCL-2 cells may have BCL-2 DLBCL cells showed an increased basal increased autophagy activity in response to autophagy autophagy activity stress compared with BCL-2 cells. To evaluate the impact of BCL-2 overexpression FL B-cells showed an increased expression of on autophagy in human lymphoma, we first compared the autophagy-related genes autophagy status of the BCL-2 Su-DHL4 with the BCL- 2 Su-DHL8 DLBCL cell lines using Western blotting and We next evaluated autophagy gene expression levels the RT Profiler PCR array (Figure 1). Overexpression in primary FL and DLBCL samples and compared them to of BCL-2 in Su-DHL4 cells was confirmed by Western RA controls. In order to differentiate autophagy activity in blotting and there was no differential expression of lymphoma B-cells from surrounding stromal cells, tumor- Beclin-1 or Bcl-xL, another binding partner of Beclin-1, infiltrating T-cells and macrophages, B-cell subsets were between these two cell lines. SQSTM1/p62 (p62) serves isolated by flow cytometry. CD3 T-cells were excluded as a link between LC3 and ubiquitinated substrates + + + and CD10 /CD19 B-cells (FL) and CD20 B-cells resulting in these two proteins being incorporated into (DLBCL and RA) were purified from primary single cell the completed autophagosome and degraded in the suspensions. B-cell receptor (BCR) isotype restriction autolysosome [22, 23]. Both p62 and LC3-II showed + + is a hallmark of FL cells, and purified CD19 /CD10 FL increased expression in the Su-DHL8 cell line (Figure 1 B-cells were found to be either κ or λ light-chain restricted A), indicative of inhibited autophagic degradation [24], (Figure 3 A). After flow sorting, mean purities of B-cells suggesting that the autophagy flux may be inactive in this were ≥95% for all samples. BCL-2- cell line compared with the BCL-2 Su-DHL4 We analyzed the autophagy-related GEP of highly cells. Using autophagy PCR array, we detected that the Su- www.impactjournals.com/oncotarget 11654 Oncotarget Table 1: Aberrantly expressed autophagy-related genes in purified and unpurified FL and DLBCL samples. www.impactjournals.com/oncotarget 11655 Oncotarget Fold changes (F.C.) in gene expression levels of unpurified (UP) FL or DLBCL samples were compared with unpurified RA-LNs and those of purified (P) FL or DLBCL samples were compared with purified reactive B cells. Genes listed in the table are genes with expression changes greater than or less than 3 fold and those greater than or less than 2 fold with a P value <0.05. Numbers of samples: purified RA n=3; FL n=5; DLBCL n=2; unpurified RA n=8; FL=8; DLBCL n=10. Significantly increased or decreased fold changes were identified using a Mann Whitney U test. Numbers in the first column indicate the functions of these genes, according to the classification of human autophagy PCR array (Sabiosciences): (1-6) Autophagy machinery genes: (1) Genes involved in autophagic vacuole formation; (2) Gene responsible for protein targeting to membrane/vacuole; (3) Genes responsible for protein transport; (4) Genes linking autophagosome to lysosome; (5) Genes involved in protein ubiquitination; (6) Genes with protease activity; (7-10) Autophagy regulatory genes: (7) Co-regulators of autophagy and apoptosis; (8) Co-regulators of autophagy and the cell cycle; (9) Autophagy induction by intracellular pathogens; (10) Autophagy in responsible to other intracellular signals. purified and unpurified FL and DLBCL diagnostic in both purified and unpurified FL and DLBCL samples tissue biopsies and compared them with non-malignant (Table 1). To consolidate these findings, increased RA samples. The results of unsupervised hierarchical expression of BECN, MAP1LC3A, ATG4B, DRAM1 and clustering are shown for purified reactive and malignant CTSD was validated in unpurified tissue using qRT-PCR. B-cells (Figure 3 B) and unpurified tissue biopsies Results were comparable to those obtained from the PCR (Figure 3 C). Seven and two autophagy machinery genes array (Suppl Table 8). These data demonstrate that both were up-regulated in purified FL and DLBCL samples, FL and DLBCL samples aberrantly express autophagy respectively (Table 1), one of which, MAP1LC3A was genes at the basal levels. In particular, FL samples which commonly up-regulated in both FL and DLBCL purified frequently overexpress BCL-2 have increased expression B-cells. Only one gene, BNIP3, showed significantly of numerous autophagy machinery and regulatory genes. decreased expression in both FL and DLBCL B-cells. In the cohort of purified samples, two FL patients BNIP3 is a hypoxia-dependent autophagy inducer and (T1979 and T5728) with high global expression of its expression is suppressed in many types of cancer [28] autophagy genes subsequently underwent transformation but overexpressed in lung and breast carcinomas [29]. to the more aggressive DLBCL later in their clinical Gene expression patterns in both FL and DLBCL were course (Suppl Figure 4 A and Suppl Table 1). In unpurified not associated with Ann Arbor stage or international tissue biopsies, 7 of 8 FL and 1 of 10 DLBCL samples prognostic index (IPI) scores (data not shown). Among showed increased global expression of autophagy genes the 46 genes which showed increased expression in compared to RA tissues (Suppl Figure 4 B). A larger these samples, 19 genes in FL and 2 genes in DLBCL number of purified DLBCL samples will be required to were autophagy machinery genes (Figure 3 C and Table establish aberrant expression of autophagy genes in this 1) and 27 genes in FL and 3 in DLBCL were autophagy disease. regulatory genes. Both BECN1 and BCL2 genes were up-regulated in FL but not in DLBCL tissue biopsies. FL showed significantly decreased expression of Expression of two lysosomal components CTSD autophagy substrates p62 and LC3 proteins (cathepsin D) and DRAM1 (damage-regulated autophagy modulator 1) [30] was significantly up-regulated in both To establish the autophagy status of FL and DLBCL, FL and DLBCL tissue biopsies, suggesting they may be primary FL, DLBCL and RA LN tissue biopsies on TMAs expressed at higher levels in the tumor microenvironment. were stained using IHC for the autophagy substrate CDKN2A (p16), a tumor-suppressor gene, is up-regulated www.impactjournals.com/oncotarget 11656 Oncotarget proteins p62 and LC3, and autophagy initiating protein significantly decreased expression in DLBCL (Figure Beclin-1 (Figure 4A). RA and FL samples were stained 4 B, D and F). Most strikingly, all FL samples showed with CD10 antibody to distinguish follicular center B-cells consistently lower levels of p62. Expression of p62 from surrounding cells. CD10 area were classified as in DLBCL and LC3 and Beclin-1 in FL and DLBCL intra-follicular areas; CD10 areas were classified as inter- displayed a heterogeneous expression pattern. In FL, follicular areas [31] (Suppl Figure 5A). significantly decreased expression of p62, LC3 and First, protein expression in FL intra-follicular areas Beclin-1 was observed in both intra-follicular and non- was compared with RA and DLBCL whole core samples. malignant inter-follicular areas (Figure 4 C, E and G), p62, LC3 and Beclin-1 showed significantly decreased suggesting autophagy may be altered in both malignant expression in FL intra-follicular areas compared with FL cells and surrounding tumor infiltrating cells. DLBCL and RA controls. Only Beclin-1 displayed Approximately 91% FL samples were BCL-2 positive + - Figure 1: Determination of basal autophagy status in BCL-2 Su-DHL4 and BCL-2 Su-DHL8 cell lines. (A) Comparison of autophagy-related protein expression by Western blotting. 50 µg proteins were loaded onto each lane of a 12-well SDS-PAGE gel. Proteins were transferred to a PVDF membrane which was probed with primary antibody at 4°C overnight. Primary antibodies were used at a 1:2000 dilution for GAPDH and at 1:1000 for all other antibodies. Levels of protein expression were measured by densitometry. Numbers below panels of Western blots indicate the ratio of a specific protein to GAPDH. (B) Supervised hierarchical clustering of significantly differentially expressed autophagy machinery and autophagy regulation genes. Heat-map shows triplicate RQ values for Su-DHL4 and Su- DHL8 cell lines. Each column represents an mRNA/RQ value and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively. Side bars were removed for clarity. (C and D) RQ values of significantly increased or decreased autophagy machinery (C) and autophagy regulatory (D) genes differentially expressed in the Su-DHL4 cell line compared with the Su-DHL8 cell line, analyzed using student t-test (P<0.05) and represented as fold changes. www.impactjournals.com/oncotarget 11657 Oncotarget Figure 2: Inhibition or induction of the autophagic flux in Su-DHL4 and Su-DHL8 cell lines. (A) Blocking autophagic flux. Cells were incubated in normal culture medium in the presence or absence of 50 µM CQ. (B) Induction of autophagy by starvation. Cells were incubated in HBSS for up to 6 hours. Cells were collected at each indicated time point for protein extraction and Western blotting. Numbers below each band indicate ratios of specific proteins to GAPDH which were determined by densitometry. (C and D) Supervised hierarchical clustering of significantly differentially expressed autophagy related genes in Su-DHL4 (C) and Su-DHL8 (D) cells after incubation in HBSS for 6 hours. Heat-map shows triplicate RQ values for normal and HBSS cultured Su-DHL4 and Su-DHL8 cell lines. Each column represents an mRNA/RQ value and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively. (E and F) RQ values of autophagy related genes significantly differentially expressed in Su-DHL4 (E) and Su-DHL8 (F) cells after incubation in HBSS for 6 hours were analyzed by the paired student t-test (p<0.05) and represented as fold changes. Red bars indicate changes in autophagy machinery genes and black and white bars indicate changes in autophagy regulatory genes. www.impactjournals.com/oncotarget 11658 Oncotarget Figure 3: Determination of expression of autophagy related genes in purified and unpurified FL and DLBCL samples. (A) Flow sorting of B-cells. Immunophenotyping was used to isolate the B-cell population from human RA, FL and DLBCL single cell - + - + + suspensions. B-cells were identified as CD3 CD20 for RA and DLBCL samples and CD3 /CD10 /CD19 for FL samples. Purified FL B-cells were confirmed as being either κ or λ light chain restricted. (B and C) Unsupervised hierarchical clustering using autophagy-related genes expressed in purified B cells (B) and unpurified bulk biopsies (C). Five FL, 2 DLBCL and 3 RA purified samples (B) and 8 FL, 10 DLBCL and 8 RA tissue biopsies (C) were analyzed by qRT-PCR. Heat-map shows RQ values where each column represents a patient and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively; gray indicates missing data. www.impactjournals.com/oncotarget 11659 Oncotarget (>30% of stained area); DLBCL samples displayed BCL-2 levels showed a negative correlation with p62 in a heterogeneous BCL-2 expression pattern (Suppl FL (P<0.05) and positive correlation with p62, LC3 or Figure 5 B and C). These results demonstrate that FL Beclin-1 in DLBCL (P<0.01), suggesting that BCL-2 has an increased basal autophagy activity, regardless of does not play a dominant role in autophagy status in these overexpression of BCL-2. lymphomas. Expression levels of p62 and LC3 showed In order to understand the association between strong positive correlation (P<0.0001) in RA, FL and BCL-2 and p62, LC3 or Beclin-1, correlations between DLBCL samples, indicating that p62 or LC3, individually these proteins in RA, FL and DLBCL were analyzed by or in combination, can be used as a marker for evaluating Pearson product-moment correlation method (Figure 5). autophagy activity. Interestingly, Beclin-1 expression Figure 4: Comparison of p62, LC3 and Beclin-1 protein expression in FL and DLBCL with RA-LN. (A) Representative histochemical stained images of p62, LC3, and Beclin-1 in RA, FL and DLBCL. Proteins were stained with polyclonal anti-p62, polyclonal anti-LC3B antibody, or a mouse anti-Beclin-1 antibody respectively. Detailed information of antibodies and their dilution are listed in the Suppl Table 5. Red arrows indicate positive cells. (B-G) Statistical analysis p62 (B and C), LC3B (D and E) and Beclin-1 (F and G) expression. All data presented are medians with interquartile ranges. Sample numbers for RA and FL in C, E and G are as same as listed in B, D and F. Statistical difference between samples was analyzed by unpaired Mann-Whitney U test. *P<0.05, **P<0.01, and ***P<0.0001. www.impactjournals.com/oncotarget 11660 Oncotarget levels were positively correlated with levels of p62 and of cathepsin D and TGM2 proteins in previously un- LC3 in both FL (P<0.01) and DLBCL (P <0.0001), treated FL, DLBCL and RA tissue biopsies and the suggesting that Beclin-1 levels is not positively associated results were reviewed by expert histopathologist (MC and with autophagy activity. RC). The expression pattern of cathepsin D and TGM2 suggested that they are not expressed by lymphoma cells, but rather macrophages (Figure 5 A). To confirm cells Cathepsin D and tissue transglutaminase-2 are highly expressing cathepsin D and/or TGM2 are tumor highly expressed in the tissue macrophages infiltrating cells, DLBCL and RA TMAs were stained with CD68, a marker for tumor-associated macrophages Gene expression profiling of DLBCL malignant (TAMs) [33, 34] (Figure 5 B). These data confirmed that B-cells may be affected by the presence of stromal cells cells expressing cathepsin D and/or TGM2 are indeed [32]. CTSD showed significantly increased expression macrophages, although co-localization between these at the gene level in FL and DLBCL sample biopsies but proteins and CD68 was not evaluated. Cathepsin D not in purified B-cells. Increased expression of TGM2 expression was significantly lower in FL but significantly (Tissue transglutaminase-2) was detected in both purified higher in DLBCL samples compared with RA controls and unpurified DLBCL samples (Table 1). To distinguish (Figure 5 C), while both FL and DLBCL samples showed the origins of these components, we evaluated expression decreased expression of TGM2 (Figure 5 D). Neither Figure 5: Multiple analysis of correlation between BCL-2, p62, LC3 and Beclin-1. (A) RA; (B) FL and (C) DLBCL. Correlation was analyzed by Pearson product-moment correlation coefficient test. ‘γ’ indicates correlation coefficient. Sample numbers, RA=30; FL=50 and DLBCL=109. www.impactjournals.com/oncotarget 11661 Oncotarget cathepsin D nor TGM2 expression was correlated with signature of malignant B-cells when analyzing autophagy autophagy status in FL or DLBCL (data not shown). in unpurified tissue biopsies by PCR array. Both cathepsin D and TGM2 showed strong positive correlation (P<0.0001) with CD68 (Supp Figure 6 A DISCUSSION and B). Expression of cathepsin D and TGM2 were also strongly correlated (P<0.0001) (Supp Figure 6 C). These Here we report that FL, an indolent NHL which results suggest that higher expression of autophagy frequently overexpresses the anti-apoptotic protein genes in TAMs may lead to misinterpretation of the gene BCL-2, showed significantly increased expression of Figure 6: Immuno-histochemical staining of cathepsin D, TGM2 and CD68. (A) Representative images of cathepsin D and TGM2 expression in RA, FL and DLBCL. (B) Representative images of CD68 expression in RA and DLBCL. Antibody dilutions were 1:1000, 1:750, or 1:8000 for anti-cathepsin D, anti-TGM2, or anti-CD68 antibodies, respectively. Images were taken with a Leixa DM2500 microscope: original magnification X200. Cathepsin D, TGM2 and CD68 protein expression levels were defined as % stained viable tissue area. (C and D) Statistical analysis of protein levels of cathepsin D (B) and TGM2 (C) were calculated as the percent stained area of viable tissue. Data collected for RA and DLBCL were from whole cores and for FL were from the CD10 intra-follicular area. Significantly increased or decreased expression between samples was analyzed by the Mann-Whitney U test. Numbers of samples used for analysis were indicated. www.impactjournals.com/oncotarget 11662 Oncotarget key autophagy genes and decreased levels of autophagy activity at the gene level. We have also confirmed by substrate protein p62 and LC3 compared with RA B-cell decreased expression of autophagy substrate proteins p62 controls. Whereas, the autophagy-related GEP and levels and LC3 in FL tissue specimens using TMAs and IHC, of autophagy substrate p62 and LC3 proteins in DLBCL, demonstrating an active autophagy at the protein levels in an aggressive NHL, was more similar to those of RA FL. Nevertheless, there is no strong and clear correlation B-cell controls. A constitutive basal level of autophagy between BCL-2 expression levels and autophagy activity. in normal tissue provides an important homeostatic, We therefore propose that autophagy activity in lymphoma housekeeping function to survive stress, such as nutrient samples may not be controlled by BCL-2. deprivation [21, 35]. The role of autophagy in cancer is FL frequently transforms to the more aggressive complex as it can prevent tumor initiation by suppressing DLBCL. Our data showed that only one gene, chronic tissue damage, inflammation, and genome MAP1LC3A, was up-regulated in DLBCL B-cells. It was instability via its quality control function or it can sustain recently reported that autophagy is positively regulated by tumor metabolism, growth, and survival via nutrient LITAF which is silenced by promoter hypermethylation recycling [36], suggesting a link between dysregulated in germinal center-derived B-cell lymphomas, suggesting autophagy and tumor progression. that autophagy may be inhibited in these lymphomas Higher expression of the anti-apoptotic protein [38]. We found that levels of p62 and LC3 proteins BCL-2 is more common in B-cell NHL than T-cell NHL showed a heterogeneous expression pattern in DLBCL and is heterogeneously expressed among the different and had no significant difference compared with RA histological subtypes [37]. Although BCL-2 is a well- controls. In addition, expression levels of p62 and LC3 established anti-apoptotic protein, it was also proposed did not show association with clinical outcomes of to suppress autophagy by binding and inhibiting Beclin-1 patients with FL (data not shown). This suggests that up- in a cardiac BCL-2 transgenic mice model [16]. A recent regulated autophagy in FL may not be associated with finding demonstrated that BCL-2 does not bind directly to transformation to DLBCL. Instead, active autophagy in Beclin-1 but instead regulate autophagy by inhibiting Bax/ FL may even suppress tumor progression by eliminating Bak mediated apoptosis [17]. We therefore determined damaged organelles and controlling genetic instability. the role of BCL-2 on autophagy in BCL-2 and BCL- Increased expression of autophagy-related genes 2 cell lines and human primary lymphoma samples. was more readily detected in FL bulk tissue biopsies. Higher levels of autophagy machinery genes were found Similar to FL purified B-cells, FL unpurified tissue also + - in BCL-2 cells compared with the BCL-2 cell line. showed up-regulation of autophagy machinery genes, + - BCL-2 and BCL-2 DLBCL cell lines showed similar including ATG16L1, MAP1LC3A, ATG9, LAMP1 and autophagy flux activity upon either autophagy inhibition HDAC6; many other autophagy machinery and regulatory by CQ or autophagy induction by nutrient-deprivation, genes were also significantly up-regulated. Among the evidenced by accumulation or degradation of p62 and LC3 up-regulated autophagy regulatory genes, TP53, MAPK8, proteins, respectively. Interestingly, the BCL-2 cell line HDAC1, DAPK1, CDKN1B, CDKN2A, UVRAG, and up-regulated more autophagy-related genes in response RPS6KB1 are positive regulators of autophagy, whereas to starvation compared to BCL-2 DLBCL cells. These AKT1, PIK3CG, BCL-2, BCL-2L1, mTOR, EIF4G1, results demonstrate that the autophagic flux is not altered and MAPK14 are negative autophagy regulators. It is by the BCL-2 protein in human malignant B-cells. In fact, obvious that more aberrantly expressed autophagy- BCL-2 cells up-regulate more autophagy-related genes related genes were detected in unpurified FL samples than BCL-2 DLBCL cells in both the resting state and in compared with purified B-cells. Our previous studies response to autophagy induction. show that FL tissues have increased numbers of CD163 As expected, the BCL-2 gene was highly expressed infiltrating macrophages [39] and CD4 T-cells [40] in the in both purified FL B-cells and unpurified tissue biopsies microenvironment. This suggests that autophagy activity compared with RA and DLBCL primary samples. To might also be altered in FL tumor infiltrating cells and identify expression signatures of autophagy-associated this is being actively explored. Notably, we did not find genes in the malignant population specifically, B-cells evidence of upregulation of autophagy related genes in our were purified from FL and DLBCL tissue biopsies by previous studies of GEP of the tumor infiltrating T-cells flow sorting. Normal B-cells were also purified from in FL [31]. RA-LNs which served as controls. Seven autophagy Fewer autophagy-related genes had altered machinery genes were up-regulated in purified FL B-cells, expression levels in DLBCL, regardless of sample including ATG9A, ATG16L1, MAP1LC3A, GABARAPL1 purification. Significantly up-regulated genes in DLBCL and ULK1, which are involved in autophagosome samples included CTSD, DRAM1 and TGM2. CTSD formation and protein transport; LAMP1, involved in and DRAM1 are lysosomal proteins which regulate the autolysosome formation; and HDAC6, involved in protein autophagic flux through the lysosome [30, 41], while ubiquitination. This result demonstrates that FL B-cells, TGM2 is involved in autophagy-dependent clearance which overexpress BCL-2 up-regulate the basal autophagy of ubiquitinated proteins [42]. We therefore tested the www.impactjournals.com/oncotarget 11663 Oncotarget origin of cells expressing high levels of cathepsin D and Human samples and ethical considerations TGM2 using TMAs. Expression of both cathepsin D and TGM2 proteins was significantly lower in FL samples but Ethical approval for the human biological materials significantly higher in DLBCL samples compared with RA used in this study was obtained in accordance with the controls. Morphological features of cells expressing higher requirements of the East London and the City Health cathepsin D or TGM2 were identical to CD68-expressing Authority Local Research Ethics Committee (Ref. No. TAMs. Indeed, expression levels of both cathepsin D 10/H0704/65). All samples were obtained from patients and TGM2 were strongly correlated to CD68 expression by informed consent. Patients selected presented at St. levels in DLBCL. We found that increased cathepsin D Bartholomew’s hospital between the years 1970-2012. expression was associated with a shorter overall survival For RT Profiler PCR array analysis, lymph node (LN) of DLBCL patients (data not shown), in agreement with biopsies and cryopreserved single cell suspensions were a previous report by Nicotra et al [43]. TGM2 has been obtained from diagnostic, previously un-treated FL (n=13) reported as a marker for progression and therapeutic and DLBCL (n=11) patients (Suppl Table 1 and 2); RA- intervention in colorectal cancer and non-small cell lung LNs (n=11) were used as controls. For tissue microarrays cancer.[44, 45] However, the role of TGM2 in DLBCL (TMAs), LN biopsies from 128 FL and 144 DLBCL is unknown. We found that TGM2 expression is not patients at diagnosis for whom quality formalin fixed associated with shorter overall survival or other prognostic paraffin embedded tissue, clinical and follow-up data markers in DLBCL (data not shown). Nevertheless, were available, as well as 28 reactive LN biopsies, were CTSD and TGM2 expression levels in DLBCL did not included on the TMAs (Suppl Table 3 and 4). reflect their expression in malignant B-cells. We therefore propose that determination of autophagy-related gene Reagents expression using unpurified lymphoma specimens could be distorted by high lysosome-containing TAMs. In summary, the role of BCL-2 in autophagy RT Profiler Human Autophagy PCR Array is currently elusive. Using the RT Profiler Human (PAHS084ZE), RNeasy mini kit, RT First Strand Autophagy PCR array and TMAs, we demonstrate Kit, primers for qPCR validation, including BECN that basal autophagy activity was up-regulated in (PPH05670B), MAP1LC3A (PPH19436A), ATG4B primary BCL-2 overexpressing FL B-cells and their (PPH15916A), DRAM1 (PPH19768F), CTSD microenviromental cells. A greater number of autophagy (PPH00112F) and RPLPO (PPH21138F) were purchased machinery genes were up-regulated in the BCL-2 DLBCL from Qiagen-Sabiosciences. 3,3’-diaminobenzidine cell line Su-DHL4 at the basal level and in response to (DAB) was from BioGenex. TRIzol® reagent, 4-12% stress, indicating that overexpression of BCL-2 does not NuPAGE gels and Hanks balanced salt solution (HBSS) inhibit the autophagic flux. Instead, inhibition of apoptosis were from Invitrogen. Chloroquine (CQ), 4’,6-diamidino- by overexpression of BCL-2 may switch on autophagy 2-phenylindole (DAPI), and all other chemicals used were in the cell in favor of eliminating aged organelles and from Sigma. Antibodies used in this study are listed in damaged proteins. However, the mechanism by which FL Suppl Table 5. cells up-regulate autophagy-related gene expression is not clear and may be due to multiple factors. We therefore Purification of B-cells by flow cytometry sorting propose that overexpression of the anti-apoptotic protein BCL-2 does not suppress autophagy activity in human FL. Primary single cell suspensions (10 cells/ml) were washed once with washing buffer containing 2% FCS in MATERIALS AND METHODS PBS (phosphate buffered saline). Non-specific bindings were blocked by incubating cells with 2% human anti-γ- globulin antibody for 30 min at 4°C. Cells were stained Cell lines and cell culture with conjugated anti-CD3/anti-CD20 or anti-CD3/ anti-CD19/anti-CD10 antibodies for 30 min at 4°C and subsequently washed once with washing buffer. Cells were Human DLBCL cell lines Su-DHL4 (BCL-2 ) and resuspended in blocking buffer containing DAPI (50ng/ Su-DHL8 (BCL-2 ) [46] were used in this study. Cells ml) and sorted on a BD FACSAria II Cell Sorter. DAPI were cultured in RPMI-1640 medium supplemented was used to discriminate live and dead cells. Following with 10% heat-inactivated fetal calf serum (FCS), 25mM selection of DAPI negative cells, T-cells were excluded by HEPES, and 2.0mM L-glutamine at 37°C in a 5% CO gating on CD3 negative (CD3 ) cells. B-cells were isolated humidified incubator. To induce autophagy, cells were - - from the DAPI /CD3 population based on expression cultured in HBSS for up to 6 hours. of B-cell markers. DLBCL and reactive B-cells were identified by CD20 expression while FL B-cells were www.impactjournals.com/oncotarget 11664 Oncotarget isolated based on dual expression of CD19 and CD10. stipulated conditions. All genes validated, including the FL B-cells were further confirmed as the malignant cell housekeeping gene RPLPO, were analyzed in triplicate population by demonstrating kappa/lambda (κ/λ) light and a no-template control (NTC) also included per gene. chain restriction [47]. Primers used in validation experiments were identical to those present on the RT Profiler Human Autophagy PCR Array. RNA extraction and cDNA conversion Tissue microarray (TMAs) and IHC analysis Total RNA was isolated from purified single B-cell suspensions or solid LN tissue biopsies with TRIzol® and/ or the RNeasy mini kit. RNA quality was assessed using TMAs were constructed using a semi-automated the Agilent 2100 Bioanalyzer (Agilent Technologies) and arraying system (TMABooster-Alphelys). Sections of a Nanodrop spectrophotometer (Thermo-Scientific); all biopsy material were stained with hemotoxylin and eosin samples had an RNA integrity number (RIN) greater than and reviewed by an expert histopathologist (C.M.) who 6 and 260/280 ratios higher than 1.9. RNA (300ng) was identified areas rich in malignant cells. Triplicate 1mm converted to cDNA by RT-PCR using the RT First Strand cores were then taken from these areas, arrayed and Kit. stained as previously described [31, 33, 34]. Slides were digitalized and image analysis was performed using a digital pathology system (Ariol, Lieca Microsystems). RT2 Profiler PCR array for detection of The Ariol image analysis classifier was trained based on expression of autophagy related genes the hue, saturation and intensity of DAB staining, such that only areas stained above a pre-determined threshold The RT Profiler PCR Array combines qRT- representing the highest intensity staining were classed PCR technology with a microarray format to allow the as positive, and lower level background expression was simultaneous detection of multiple gene expression levels excluded. A further classifier was used to determine the in a rapid manner. The RT Profiler Human Autophagy total viable tissue area. Combining these two classifiers, PCR Array contains primers against 84 genes involved in the percent stained area was calculated. In FL cores, different stages of the autophagy pathway (Suppl Table intra-follicular areas were selected based on expression 6 and 7) as well as primers against five housekeeping of CD10 and clear follicle morphology. Results were genes which are used for data normalization. According manually and blindly reviewed and reported as an average to the manufacturer’s protocol, qRT-PCR was performed of the triplicate cores. Protein levels were expressed as % by adding 2.8ng cDNA mixed with RT SYBR Green stained area [33]. Mastermix (Sabiosiciences) to each well which already contained primers directed against the gene of interest. Western blotting mRNA levels were analyzed using the ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems) and a Proteins were extracted with lysis buffer and 50 µg dissociation curve analysis step included to verify PCR of proteins added to each lane of 4-12% NuPAGE gels and specificity. Relative quantity (RQ) values were calculated -∆∆C Western blotting was performed as previously described using the formula RQ = 2 . Target cycle threshold (C ) T T [33]. values were normalized to the housekeeping gene RPLPO generating a ∆C value. The average ∆C of reactive T T controls was used as the calibrator sample on a per gene Statistical analysis basis and was subtracted from each ∆C generating a delta ∆C value which was linearized by raising to the power of Statistical analysis was performed using GraphPad -∆∆C 2 (2 ). Genes with a fold change ≥3 or ≤-3 or a FC ≥2 Prism software (version 5.03). Data are shown as either or ≤-2 and a p value <0.05 using a Mann-Whitney U test mean ± SD or median with interquartile range when or student t-test were taken to be biologically meaningful. variation was high. Significant differences between groups Hierarchical clustering was performed using Euclidean with unequal size were analyzed with the Mann-Whitney distance measure and an average agglomeration available U test and those with equal size were analyzed using the within the R statistical computing environment. student t-test. Pearson product-moment correlation method was used to analyze linear correlation between two groups. qRT-PCR validation All P-values less than 0.05 were considered statistically significant. Following conversion of RNA (500ng) to cDNA, samples were prepared for qRT-PCR as previously described and the qRT-PCR assay run under the previously www.impactjournals.com/oncotarget 11665 Oncotarget therapy. Oncogene. 2012; 31(8):939-953. Conflict of interest 6. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C, Dipaola The authors declare that they have no conflict of RS, Karantza-Wadsworth V and White E. Autophagy interest. suppresses tumorigenesis through elimination of p62. Cell. 2009; 137(6):1062-1075. Editorial note 7. Jia L, Gopinathan G, Sukumar JT and Gribben JG. Blocking Autophagy Prevents Bortezomib-Induced NF-kappaB This paper has been accepted based in part on peer- Activation by Reducing I-kappaBalpha Degradation in review conducted by another journal and the authors’ Lymphoma Cells. PLoS One. 2012; 7(2):e32584. response and revisions as well as expedited peer-review 8. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson in Oncotarget. D, Chen G, Mukherjee C, Shi Y, Gelinas C, Fan Y, Nelson DA, Jin S and White E. Autophagy promotes tumor Authors’ contribution cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006; 10(1):51-64. 9. Yang ZJ, Chee CE, Huang S and Sinicrope FA. The role of L.J and J.G.G contributed project design and autophagy in cancer: therapeutic implications. Mol Cancer manuscript writing. A.M, A.C, and R.C performed Ther. 2011; 10(9):1533-1541. experiments. A.M, L.J, J.M, and R.D.P contributed data collection and analysis. J.M and A.W provided 10. Vogl DT, Stadtmauer EA, Tan KS, Heitjan DF, Davis LE, clinical information of CLL patients. M.C, R.C and A.C Pontiggia L, Rangwala R, Piao S, Chang YC, Scott EC, contributed histopathological identification of lymphoma Paul TM, Nichols CW, Porter DL, Kaplan J, Mallon G, samples. S.I provided excellent tissue bank service. All Bradner JE, et al. Combined autophagy and proteasome authors contributed manuscript writing. inhibition: A phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma. Autophagy. 2014; 10(8):1380-1390. ACKNOWLEDGEMENTS 11. Lamoureux F, Thomas C, Crafter C, Kumano M, Zhang This project is funded by CRUK PhD studentship to F, Davies BR, Gleave ME and Zoubeidi A. Blocked L.J for supporting A.M. and a program grant P01 CA81538 autophagy using lysosomotropic agents sensitizes resistant from the National Cancer Institute to the CLL Research prostate tumor cells to the novel Akt inhibitor AZD5363. Consortium to JGG. 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Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2

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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 22 Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2 1 2 1 1 1 Aine McCarthy , Jacek Marzec , Andrew Clear , Robert D. Petty , Rita Coutinho , 1 1 1 1 Janet Matthews , Andrew Wilson , Sameena Iqbal , Maria Calaminici , John G. 1 1 Gribben and Li Jia Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Centre for Molecular Oncology2, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Correspondence to: Li Jia, email: L.jia@qmul.ac.uk Keywords: Autophagy, BCL-2, follicular lymphoma, autophagy PCR array, and tissue microarray Received: October 09, 2014 Accepted: October 18, 2014 Published: October 18, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL- + - 2 /BCL-2 lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the + - BCL-2 Su-DHL4 cell line compared with BCL-2 Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression proles fi were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease. and inhibition of autophagy can enhance tumor cell death INTRODUCTION by diverse anticancer therapies [9-11]. Follicular lymphoma (FL) is the second most Macroautophagy (hereafter referred to as common lymphoma diagnosed in the United States and autophagy) is a physical and pathological process that Western Europe, accounting for approximately 20% of all allows eukaryotic cells sequester portions of cytoplasm non-Hodgkin lymphomas (NHLs) and 70% of indolent to form autophagosomes and target them for degradation lymphomas and is generally considered incurable [12, through the fusion of autophagosomes with lysosomes 13]. The t(14;18)(q32;q21) translocation characterizes where they are degraded and recycled [1-4]. Growing approximately 85% of FL and 20% of diffuse large evidence demonstrates that autophagy plays important and B-cell lymphoma (DLBCL) and results in constitutive paradoxical roles in tumorigenesis and in the treatment of overexpression of the anti-apoptotic protein BCL-2 [14, cancer [4-7]. Autophagy can suppress tumorigenesis by 15]. Overexpression of BCL-2 in NHLs plays important removing damaged organelles/proteins and limiting cell roles in disease pathogenesis and resistance to apoptosis. growth and genomic instability [6]. In contrast, induction It is currently unknown whether BCL-2 plays an important of autophagy by metabolic stress in apoptosis deficient role in regulation of autophagy in indolent FL and more tumor cells can support tumor cell survival [8]. Abundant aggressive DLBCL. preclinical evidence indicates that stress-induced The role of the anti-apoptotic protein BCL-2 in autophagy in tumor cells is predominantly cytoprotective www.impactjournals.com/oncotarget 11653 Oncotarget autophagy is currently debated and remains unclear. DHL4 cell line was also distinguishable from Su-DHL8 BCL-2 has been proposed as a major binding partner of cells based on its autophagy-related gene expression Beclin-1 which it binds in a nutrient-dependent manner, profile (GEP) (Figure 1 B and Suppl Figure 1). Four consequently down-regulating levels of starvation- autophagy machinery and nine autophagy regulatory genes mediated autophagy [16]. Recently, it was reported that were up-regulated in the Su-DHL4 cell line compared to the anti-apoptotic BCL-2 family members do not directly Su-DHL8 cells (Figure 1 C and D). These data support inhibit components of the autophagic pathway but instead that BCL-2 Su-DHL4 cells may have higher basal level affect autophagy indirectly, owing to their inhibition of autophagy activity compared with BCL-2 Su-DHL8 cells. Bax and Bak [17]. Autophagy is often activated as an We next sought to determine the capacity of + - adaptive response against ER stress [18, 19]. In cancer the autophagic flux in both BCL-2 and BCL-2 cells. cells, metabolic stress strongly induces autophagy which is Inhibition of the autophagic flux by treatment of cells sustained when apoptosis is blocked [8, 20, 21]. Questions with CQ [7, 25] or induction of autophagy by nutrient raised over the roles of BCL-2 in autophagy are: whether deprivation led to increased or decreased p62 and LC3B-I/ overexpression of BCL-2 in human tumors inhibits both LC3B-II protein expression respectively in both cell apoptosis and autophagy; and whether inhibition of lines (Figure 2 A and B), demonstrating that autophagy apoptosis by overexpression of BCL-2 could activate activity was not suppressed in the BCL-2 Su-DHL4 cells. the autophagic pathway in favor of prolonged tumor cell Beclin-1 expression did not change in response to either survival. treatment, indicating that its levels are not governed by The role of BCL-2 in autophagy in human NHLs the autophagy flux. Both Su-DHL4 (Figure 2 C and E has not previously been reported. We hypothesize that and Suppl Figure 2) and Su-DHL8 (Figure 2 D and F and overexpression of BCL-2 in FL may cause an increased Suppl Figure 3) cell lines responded to nutrient deprivation autophagy activity due to suppression of apoptosis. In this by up-regulating and down-regulating autophagy-related article, we aimed to determine whether overexpression genes. Among them, SQSTM1 (p62) and CDKN1B genes of BCL-2 could alter autophagy status in BCL-2 positive showed significantly increased expression in both cell + - (BCL-2 ) and negative (BCL-2 ) DLBCL cell lines, lines (Figure 2 E and F). Between 2 and 6 hours starvation, primary FL, DLBCL and reactive (RA) samples using we also noted increased p62 protein expression despite both autophagy RT Profiler PCR Array and tissue increased autophagic degradation (Figure 2 B). A similar microarray (TMA). We demonstrate for the first time that phenomenon has also been observed in mouse embryonic overexpression of BCL-2 does not inhibit autophagy in fibroblasts [26]. Increased expression of CDKN1B in human FL. response to starvation may cause cell cycle arrest in the G1 phase [27]. In addition, the Su-DHL4 cell line also showed significantly increased expression of key RESULTS autophagy machinery genes including GABARAPL1, GABARAPL2, MAP1LC3B (LC3B) and CTSS (Figure 2 C + and E). These results suggest that BCL-2 cells may have BCL-2 DLBCL cells showed an increased basal increased autophagy activity in response to autophagy autophagy activity stress compared with BCL-2 cells. To evaluate the impact of BCL-2 overexpression FL B-cells showed an increased expression of on autophagy in human lymphoma, we first compared the autophagy-related genes autophagy status of the BCL-2 Su-DHL4 with the BCL- 2 Su-DHL8 DLBCL cell lines using Western blotting and We next evaluated autophagy gene expression levels the RT Profiler PCR array (Figure 1). Overexpression in primary FL and DLBCL samples and compared them to of BCL-2 in Su-DHL4 cells was confirmed by Western RA controls. In order to differentiate autophagy activity in blotting and there was no differential expression of lymphoma B-cells from surrounding stromal cells, tumor- Beclin-1 or Bcl-xL, another binding partner of Beclin-1, infiltrating T-cells and macrophages, B-cell subsets were between these two cell lines. SQSTM1/p62 (p62) serves isolated by flow cytometry. CD3 T-cells were excluded as a link between LC3 and ubiquitinated substrates + + + and CD10 /CD19 B-cells (FL) and CD20 B-cells resulting in these two proteins being incorporated into (DLBCL and RA) were purified from primary single cell the completed autophagosome and degraded in the suspensions. B-cell receptor (BCR) isotype restriction autolysosome [22, 23]. Both p62 and LC3-II showed + + is a hallmark of FL cells, and purified CD19 /CD10 FL increased expression in the Su-DHL8 cell line (Figure 1 B-cells were found to be either κ or λ light-chain restricted A), indicative of inhibited autophagic degradation [24], (Figure 3 A). After flow sorting, mean purities of B-cells suggesting that the autophagy flux may be inactive in this were ≥95% for all samples. BCL-2- cell line compared with the BCL-2 Su-DHL4 We analyzed the autophagy-related GEP of highly cells. Using autophagy PCR array, we detected that the Su- www.impactjournals.com/oncotarget 11654 Oncotarget Table 1: Aberrantly expressed autophagy-related genes in purified and unpurified FL and DLBCL samples. www.impactjournals.com/oncotarget 11655 Oncotarget Fold changes (F.C.) in gene expression levels of unpurified (UP) FL or DLBCL samples were compared with unpurified RA-LNs and those of purified (P) FL or DLBCL samples were compared with purified reactive B cells. Genes listed in the table are genes with expression changes greater than or less than 3 fold and those greater than or less than 2 fold with a P value <0.05. Numbers of samples: purified RA n=3; FL n=5; DLBCL n=2; unpurified RA n=8; FL=8; DLBCL n=10. Significantly increased or decreased fold changes were identified using a Mann Whitney U test. Numbers in the first column indicate the functions of these genes, according to the classification of human autophagy PCR array (Sabiosciences): (1-6) Autophagy machinery genes: (1) Genes involved in autophagic vacuole formation; (2) Gene responsible for protein targeting to membrane/vacuole; (3) Genes responsible for protein transport; (4) Genes linking autophagosome to lysosome; (5) Genes involved in protein ubiquitination; (6) Genes with protease activity; (7-10) Autophagy regulatory genes: (7) Co-regulators of autophagy and apoptosis; (8) Co-regulators of autophagy and the cell cycle; (9) Autophagy induction by intracellular pathogens; (10) Autophagy in responsible to other intracellular signals. purified and unpurified FL and DLBCL diagnostic in both purified and unpurified FL and DLBCL samples tissue biopsies and compared them with non-malignant (Table 1). To consolidate these findings, increased RA samples. The results of unsupervised hierarchical expression of BECN, MAP1LC3A, ATG4B, DRAM1 and clustering are shown for purified reactive and malignant CTSD was validated in unpurified tissue using qRT-PCR. B-cells (Figure 3 B) and unpurified tissue biopsies Results were comparable to those obtained from the PCR (Figure 3 C). Seven and two autophagy machinery genes array (Suppl Table 8). These data demonstrate that both were up-regulated in purified FL and DLBCL samples, FL and DLBCL samples aberrantly express autophagy respectively (Table 1), one of which, MAP1LC3A was genes at the basal levels. In particular, FL samples which commonly up-regulated in both FL and DLBCL purified frequently overexpress BCL-2 have increased expression B-cells. Only one gene, BNIP3, showed significantly of numerous autophagy machinery and regulatory genes. decreased expression in both FL and DLBCL B-cells. In the cohort of purified samples, two FL patients BNIP3 is a hypoxia-dependent autophagy inducer and (T1979 and T5728) with high global expression of its expression is suppressed in many types of cancer [28] autophagy genes subsequently underwent transformation but overexpressed in lung and breast carcinomas [29]. to the more aggressive DLBCL later in their clinical Gene expression patterns in both FL and DLBCL were course (Suppl Figure 4 A and Suppl Table 1). In unpurified not associated with Ann Arbor stage or international tissue biopsies, 7 of 8 FL and 1 of 10 DLBCL samples prognostic index (IPI) scores (data not shown). Among showed increased global expression of autophagy genes the 46 genes which showed increased expression in compared to RA tissues (Suppl Figure 4 B). A larger these samples, 19 genes in FL and 2 genes in DLBCL number of purified DLBCL samples will be required to were autophagy machinery genes (Figure 3 C and Table establish aberrant expression of autophagy genes in this 1) and 27 genes in FL and 3 in DLBCL were autophagy disease. regulatory genes. Both BECN1 and BCL2 genes were up-regulated in FL but not in DLBCL tissue biopsies. FL showed significantly decreased expression of Expression of two lysosomal components CTSD autophagy substrates p62 and LC3 proteins (cathepsin D) and DRAM1 (damage-regulated autophagy modulator 1) [30] was significantly up-regulated in both To establish the autophagy status of FL and DLBCL, FL and DLBCL tissue biopsies, suggesting they may be primary FL, DLBCL and RA LN tissue biopsies on TMAs expressed at higher levels in the tumor microenvironment. were stained using IHC for the autophagy substrate CDKN2A (p16), a tumor-suppressor gene, is up-regulated www.impactjournals.com/oncotarget 11656 Oncotarget proteins p62 and LC3, and autophagy initiating protein significantly decreased expression in DLBCL (Figure Beclin-1 (Figure 4A). RA and FL samples were stained 4 B, D and F). Most strikingly, all FL samples showed with CD10 antibody to distinguish follicular center B-cells consistently lower levels of p62. Expression of p62 from surrounding cells. CD10 area were classified as in DLBCL and LC3 and Beclin-1 in FL and DLBCL intra-follicular areas; CD10 areas were classified as inter- displayed a heterogeneous expression pattern. In FL, follicular areas [31] (Suppl Figure 5A). significantly decreased expression of p62, LC3 and First, protein expression in FL intra-follicular areas Beclin-1 was observed in both intra-follicular and non- was compared with RA and DLBCL whole core samples. malignant inter-follicular areas (Figure 4 C, E and G), p62, LC3 and Beclin-1 showed significantly decreased suggesting autophagy may be altered in both malignant expression in FL intra-follicular areas compared with FL cells and surrounding tumor infiltrating cells. DLBCL and RA controls. Only Beclin-1 displayed Approximately 91% FL samples were BCL-2 positive + - Figure 1: Determination of basal autophagy status in BCL-2 Su-DHL4 and BCL-2 Su-DHL8 cell lines. (A) Comparison of autophagy-related protein expression by Western blotting. 50 µg proteins were loaded onto each lane of a 12-well SDS-PAGE gel. Proteins were transferred to a PVDF membrane which was probed with primary antibody at 4°C overnight. Primary antibodies were used at a 1:2000 dilution for GAPDH and at 1:1000 for all other antibodies. Levels of protein expression were measured by densitometry. Numbers below panels of Western blots indicate the ratio of a specific protein to GAPDH. (B) Supervised hierarchical clustering of significantly differentially expressed autophagy machinery and autophagy regulation genes. Heat-map shows triplicate RQ values for Su-DHL4 and Su- DHL8 cell lines. Each column represents an mRNA/RQ value and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively. Side bars were removed for clarity. (C and D) RQ values of significantly increased or decreased autophagy machinery (C) and autophagy regulatory (D) genes differentially expressed in the Su-DHL4 cell line compared with the Su-DHL8 cell line, analyzed using student t-test (P<0.05) and represented as fold changes. www.impactjournals.com/oncotarget 11657 Oncotarget Figure 2: Inhibition or induction of the autophagic flux in Su-DHL4 and Su-DHL8 cell lines. (A) Blocking autophagic flux. Cells were incubated in normal culture medium in the presence or absence of 50 µM CQ. (B) Induction of autophagy by starvation. Cells were incubated in HBSS for up to 6 hours. Cells were collected at each indicated time point for protein extraction and Western blotting. Numbers below each band indicate ratios of specific proteins to GAPDH which were determined by densitometry. (C and D) Supervised hierarchical clustering of significantly differentially expressed autophagy related genes in Su-DHL4 (C) and Su-DHL8 (D) cells after incubation in HBSS for 6 hours. Heat-map shows triplicate RQ values for normal and HBSS cultured Su-DHL4 and Su-DHL8 cell lines. Each column represents an mRNA/RQ value and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively. (E and F) RQ values of autophagy related genes significantly differentially expressed in Su-DHL4 (E) and Su-DHL8 (F) cells after incubation in HBSS for 6 hours were analyzed by the paired student t-test (p<0.05) and represented as fold changes. Red bars indicate changes in autophagy machinery genes and black and white bars indicate changes in autophagy regulatory genes. www.impactjournals.com/oncotarget 11658 Oncotarget Figure 3: Determination of expression of autophagy related genes in purified and unpurified FL and DLBCL samples. (A) Flow sorting of B-cells. Immunophenotyping was used to isolate the B-cell population from human RA, FL and DLBCL single cell - + - + + suspensions. B-cells were identified as CD3 CD20 for RA and DLBCL samples and CD3 /CD10 /CD19 for FL samples. Purified FL B-cells were confirmed as being either κ or λ light chain restricted. (B and C) Unsupervised hierarchical clustering using autophagy-related genes expressed in purified B cells (B) and unpurified bulk biopsies (C). Five FL, 2 DLBCL and 3 RA purified samples (B) and 8 FL, 10 DLBCL and 8 RA tissue biopsies (C) were analyzed by qRT-PCR. Heat-map shows RQ values where each column represents a patient and each row a gene. Gene expression levels are represented as a gradient of blue to red color indicating low and high expression respectively; gray indicates missing data. www.impactjournals.com/oncotarget 11659 Oncotarget (>30% of stained area); DLBCL samples displayed BCL-2 levels showed a negative correlation with p62 in a heterogeneous BCL-2 expression pattern (Suppl FL (P<0.05) and positive correlation with p62, LC3 or Figure 5 B and C). These results demonstrate that FL Beclin-1 in DLBCL (P<0.01), suggesting that BCL-2 has an increased basal autophagy activity, regardless of does not play a dominant role in autophagy status in these overexpression of BCL-2. lymphomas. Expression levels of p62 and LC3 showed In order to understand the association between strong positive correlation (P<0.0001) in RA, FL and BCL-2 and p62, LC3 or Beclin-1, correlations between DLBCL samples, indicating that p62 or LC3, individually these proteins in RA, FL and DLBCL were analyzed by or in combination, can be used as a marker for evaluating Pearson product-moment correlation method (Figure 5). autophagy activity. Interestingly, Beclin-1 expression Figure 4: Comparison of p62, LC3 and Beclin-1 protein expression in FL and DLBCL with RA-LN. (A) Representative histochemical stained images of p62, LC3, and Beclin-1 in RA, FL and DLBCL. Proteins were stained with polyclonal anti-p62, polyclonal anti-LC3B antibody, or a mouse anti-Beclin-1 antibody respectively. Detailed information of antibodies and their dilution are listed in the Suppl Table 5. Red arrows indicate positive cells. (B-G) Statistical analysis p62 (B and C), LC3B (D and E) and Beclin-1 (F and G) expression. All data presented are medians with interquartile ranges. Sample numbers for RA and FL in C, E and G are as same as listed in B, D and F. Statistical difference between samples was analyzed by unpaired Mann-Whitney U test. *P<0.05, **P<0.01, and ***P<0.0001. www.impactjournals.com/oncotarget 11660 Oncotarget levels were positively correlated with levels of p62 and of cathepsin D and TGM2 proteins in previously un- LC3 in both FL (P<0.01) and DLBCL (P <0.0001), treated FL, DLBCL and RA tissue biopsies and the suggesting that Beclin-1 levels is not positively associated results were reviewed by expert histopathologist (MC and with autophagy activity. RC). The expression pattern of cathepsin D and TGM2 suggested that they are not expressed by lymphoma cells, but rather macrophages (Figure 5 A). To confirm cells Cathepsin D and tissue transglutaminase-2 are highly expressing cathepsin D and/or TGM2 are tumor highly expressed in the tissue macrophages infiltrating cells, DLBCL and RA TMAs were stained with CD68, a marker for tumor-associated macrophages Gene expression profiling of DLBCL malignant (TAMs) [33, 34] (Figure 5 B). These data confirmed that B-cells may be affected by the presence of stromal cells cells expressing cathepsin D and/or TGM2 are indeed [32]. CTSD showed significantly increased expression macrophages, although co-localization between these at the gene level in FL and DLBCL sample biopsies but proteins and CD68 was not evaluated. Cathepsin D not in purified B-cells. Increased expression of TGM2 expression was significantly lower in FL but significantly (Tissue transglutaminase-2) was detected in both purified higher in DLBCL samples compared with RA controls and unpurified DLBCL samples (Table 1). To distinguish (Figure 5 C), while both FL and DLBCL samples showed the origins of these components, we evaluated expression decreased expression of TGM2 (Figure 5 D). Neither Figure 5: Multiple analysis of correlation between BCL-2, p62, LC3 and Beclin-1. (A) RA; (B) FL and (C) DLBCL. Correlation was analyzed by Pearson product-moment correlation coefficient test. ‘γ’ indicates correlation coefficient. Sample numbers, RA=30; FL=50 and DLBCL=109. www.impactjournals.com/oncotarget 11661 Oncotarget cathepsin D nor TGM2 expression was correlated with signature of malignant B-cells when analyzing autophagy autophagy status in FL or DLBCL (data not shown). in unpurified tissue biopsies by PCR array. Both cathepsin D and TGM2 showed strong positive correlation (P<0.0001) with CD68 (Supp Figure 6 A DISCUSSION and B). Expression of cathepsin D and TGM2 were also strongly correlated (P<0.0001) (Supp Figure 6 C). These Here we report that FL, an indolent NHL which results suggest that higher expression of autophagy frequently overexpresses the anti-apoptotic protein genes in TAMs may lead to misinterpretation of the gene BCL-2, showed significantly increased expression of Figure 6: Immuno-histochemical staining of cathepsin D, TGM2 and CD68. (A) Representative images of cathepsin D and TGM2 expression in RA, FL and DLBCL. (B) Representative images of CD68 expression in RA and DLBCL. Antibody dilutions were 1:1000, 1:750, or 1:8000 for anti-cathepsin D, anti-TGM2, or anti-CD68 antibodies, respectively. Images were taken with a Leixa DM2500 microscope: original magnification X200. Cathepsin D, TGM2 and CD68 protein expression levels were defined as % stained viable tissue area. (C and D) Statistical analysis of protein levels of cathepsin D (B) and TGM2 (C) were calculated as the percent stained area of viable tissue. Data collected for RA and DLBCL were from whole cores and for FL were from the CD10 intra-follicular area. Significantly increased or decreased expression between samples was analyzed by the Mann-Whitney U test. Numbers of samples used for analysis were indicated. www.impactjournals.com/oncotarget 11662 Oncotarget key autophagy genes and decreased levels of autophagy activity at the gene level. We have also confirmed by substrate protein p62 and LC3 compared with RA B-cell decreased expression of autophagy substrate proteins p62 controls. Whereas, the autophagy-related GEP and levels and LC3 in FL tissue specimens using TMAs and IHC, of autophagy substrate p62 and LC3 proteins in DLBCL, demonstrating an active autophagy at the protein levels in an aggressive NHL, was more similar to those of RA FL. Nevertheless, there is no strong and clear correlation B-cell controls. A constitutive basal level of autophagy between BCL-2 expression levels and autophagy activity. in normal tissue provides an important homeostatic, We therefore propose that autophagy activity in lymphoma housekeeping function to survive stress, such as nutrient samples may not be controlled by BCL-2. deprivation [21, 35]. The role of autophagy in cancer is FL frequently transforms to the more aggressive complex as it can prevent tumor initiation by suppressing DLBCL. Our data showed that only one gene, chronic tissue damage, inflammation, and genome MAP1LC3A, was up-regulated in DLBCL B-cells. It was instability via its quality control function or it can sustain recently reported that autophagy is positively regulated by tumor metabolism, growth, and survival via nutrient LITAF which is silenced by promoter hypermethylation recycling [36], suggesting a link between dysregulated in germinal center-derived B-cell lymphomas, suggesting autophagy and tumor progression. that autophagy may be inhibited in these lymphomas Higher expression of the anti-apoptotic protein [38]. We found that levels of p62 and LC3 proteins BCL-2 is more common in B-cell NHL than T-cell NHL showed a heterogeneous expression pattern in DLBCL and is heterogeneously expressed among the different and had no significant difference compared with RA histological subtypes [37]. Although BCL-2 is a well- controls. In addition, expression levels of p62 and LC3 established anti-apoptotic protein, it was also proposed did not show association with clinical outcomes of to suppress autophagy by binding and inhibiting Beclin-1 patients with FL (data not shown). This suggests that up- in a cardiac BCL-2 transgenic mice model [16]. A recent regulated autophagy in FL may not be associated with finding demonstrated that BCL-2 does not bind directly to transformation to DLBCL. Instead, active autophagy in Beclin-1 but instead regulate autophagy by inhibiting Bax/ FL may even suppress tumor progression by eliminating Bak mediated apoptosis [17]. We therefore determined damaged organelles and controlling genetic instability. the role of BCL-2 on autophagy in BCL-2 and BCL- Increased expression of autophagy-related genes 2 cell lines and human primary lymphoma samples. was more readily detected in FL bulk tissue biopsies. Higher levels of autophagy machinery genes were found Similar to FL purified B-cells, FL unpurified tissue also + - in BCL-2 cells compared with the BCL-2 cell line. showed up-regulation of autophagy machinery genes, + - BCL-2 and BCL-2 DLBCL cell lines showed similar including ATG16L1, MAP1LC3A, ATG9, LAMP1 and autophagy flux activity upon either autophagy inhibition HDAC6; many other autophagy machinery and regulatory by CQ or autophagy induction by nutrient-deprivation, genes were also significantly up-regulated. Among the evidenced by accumulation or degradation of p62 and LC3 up-regulated autophagy regulatory genes, TP53, MAPK8, proteins, respectively. Interestingly, the BCL-2 cell line HDAC1, DAPK1, CDKN1B, CDKN2A, UVRAG, and up-regulated more autophagy-related genes in response RPS6KB1 are positive regulators of autophagy, whereas to starvation compared to BCL-2 DLBCL cells. These AKT1, PIK3CG, BCL-2, BCL-2L1, mTOR, EIF4G1, results demonstrate that the autophagic flux is not altered and MAPK14 are negative autophagy regulators. It is by the BCL-2 protein in human malignant B-cells. In fact, obvious that more aberrantly expressed autophagy- BCL-2 cells up-regulate more autophagy-related genes related genes were detected in unpurified FL samples than BCL-2 DLBCL cells in both the resting state and in compared with purified B-cells. Our previous studies response to autophagy induction. show that FL tissues have increased numbers of CD163 As expected, the BCL-2 gene was highly expressed infiltrating macrophages [39] and CD4 T-cells [40] in the in both purified FL B-cells and unpurified tissue biopsies microenvironment. This suggests that autophagy activity compared with RA and DLBCL primary samples. To might also be altered in FL tumor infiltrating cells and identify expression signatures of autophagy-associated this is being actively explored. Notably, we did not find genes in the malignant population specifically, B-cells evidence of upregulation of autophagy related genes in our were purified from FL and DLBCL tissue biopsies by previous studies of GEP of the tumor infiltrating T-cells flow sorting. Normal B-cells were also purified from in FL [31]. RA-LNs which served as controls. Seven autophagy Fewer autophagy-related genes had altered machinery genes were up-regulated in purified FL B-cells, expression levels in DLBCL, regardless of sample including ATG9A, ATG16L1, MAP1LC3A, GABARAPL1 purification. Significantly up-regulated genes in DLBCL and ULK1, which are involved in autophagosome samples included CTSD, DRAM1 and TGM2. CTSD formation and protein transport; LAMP1, involved in and DRAM1 are lysosomal proteins which regulate the autolysosome formation; and HDAC6, involved in protein autophagic flux through the lysosome [30, 41], while ubiquitination. This result demonstrates that FL B-cells, TGM2 is involved in autophagy-dependent clearance which overexpress BCL-2 up-regulate the basal autophagy of ubiquitinated proteins [42]. We therefore tested the www.impactjournals.com/oncotarget 11663 Oncotarget origin of cells expressing high levels of cathepsin D and Human samples and ethical considerations TGM2 using TMAs. Expression of both cathepsin D and TGM2 proteins was significantly lower in FL samples but Ethical approval for the human biological materials significantly higher in DLBCL samples compared with RA used in this study was obtained in accordance with the controls. Morphological features of cells expressing higher requirements of the East London and the City Health cathepsin D or TGM2 were identical to CD68-expressing Authority Local Research Ethics Committee (Ref. No. TAMs. Indeed, expression levels of both cathepsin D 10/H0704/65). All samples were obtained from patients and TGM2 were strongly correlated to CD68 expression by informed consent. Patients selected presented at St. levels in DLBCL. We found that increased cathepsin D Bartholomew’s hospital between the years 1970-2012. expression was associated with a shorter overall survival For RT Profiler PCR array analysis, lymph node (LN) of DLBCL patients (data not shown), in agreement with biopsies and cryopreserved single cell suspensions were a previous report by Nicotra et al [43]. TGM2 has been obtained from diagnostic, previously un-treated FL (n=13) reported as a marker for progression and therapeutic and DLBCL (n=11) patients (Suppl Table 1 and 2); RA- intervention in colorectal cancer and non-small cell lung LNs (n=11) were used as controls. For tissue microarrays cancer.[44, 45] However, the role of TGM2 in DLBCL (TMAs), LN biopsies from 128 FL and 144 DLBCL is unknown. We found that TGM2 expression is not patients at diagnosis for whom quality formalin fixed associated with shorter overall survival or other prognostic paraffin embedded tissue, clinical and follow-up data markers in DLBCL (data not shown). Nevertheless, were available, as well as 28 reactive LN biopsies, were CTSD and TGM2 expression levels in DLBCL did not included on the TMAs (Suppl Table 3 and 4). reflect their expression in malignant B-cells. We therefore propose that determination of autophagy-related gene Reagents expression using unpurified lymphoma specimens could be distorted by high lysosome-containing TAMs. In summary, the role of BCL-2 in autophagy RT Profiler Human Autophagy PCR Array is currently elusive. Using the RT Profiler Human (PAHS084ZE), RNeasy mini kit, RT First Strand Autophagy PCR array and TMAs, we demonstrate Kit, primers for qPCR validation, including BECN that basal autophagy activity was up-regulated in (PPH05670B), MAP1LC3A (PPH19436A), ATG4B primary BCL-2 overexpressing FL B-cells and their (PPH15916A), DRAM1 (PPH19768F), CTSD microenviromental cells. A greater number of autophagy (PPH00112F) and RPLPO (PPH21138F) were purchased machinery genes were up-regulated in the BCL-2 DLBCL from Qiagen-Sabiosciences. 3,3’-diaminobenzidine cell line Su-DHL4 at the basal level and in response to (DAB) was from BioGenex. TRIzol® reagent, 4-12% stress, indicating that overexpression of BCL-2 does not NuPAGE gels and Hanks balanced salt solution (HBSS) inhibit the autophagic flux. Instead, inhibition of apoptosis were from Invitrogen. Chloroquine (CQ), 4’,6-diamidino- by overexpression of BCL-2 may switch on autophagy 2-phenylindole (DAPI), and all other chemicals used were in the cell in favor of eliminating aged organelles and from Sigma. Antibodies used in this study are listed in damaged proteins. However, the mechanism by which FL Suppl Table 5. cells up-regulate autophagy-related gene expression is not clear and may be due to multiple factors. We therefore Purification of B-cells by flow cytometry sorting propose that overexpression of the anti-apoptotic protein BCL-2 does not suppress autophagy activity in human FL. Primary single cell suspensions (10 cells/ml) were washed once with washing buffer containing 2% FCS in MATERIALS AND METHODS PBS (phosphate buffered saline). Non-specific bindings were blocked by incubating cells with 2% human anti-γ- globulin antibody for 30 min at 4°C. Cells were stained Cell lines and cell culture with conjugated anti-CD3/anti-CD20 or anti-CD3/ anti-CD19/anti-CD10 antibodies for 30 min at 4°C and subsequently washed once with washing buffer. Cells were Human DLBCL cell lines Su-DHL4 (BCL-2 ) and resuspended in blocking buffer containing DAPI (50ng/ Su-DHL8 (BCL-2 ) [46] were used in this study. Cells ml) and sorted on a BD FACSAria II Cell Sorter. DAPI were cultured in RPMI-1640 medium supplemented was used to discriminate live and dead cells. Following with 10% heat-inactivated fetal calf serum (FCS), 25mM selection of DAPI negative cells, T-cells were excluded by HEPES, and 2.0mM L-glutamine at 37°C in a 5% CO gating on CD3 negative (CD3 ) cells. B-cells were isolated humidified incubator. To induce autophagy, cells were - - from the DAPI /CD3 population based on expression cultured in HBSS for up to 6 hours. of B-cell markers. DLBCL and reactive B-cells were identified by CD20 expression while FL B-cells were www.impactjournals.com/oncotarget 11664 Oncotarget isolated based on dual expression of CD19 and CD10. stipulated conditions. All genes validated, including the FL B-cells were further confirmed as the malignant cell housekeeping gene RPLPO, were analyzed in triplicate population by demonstrating kappa/lambda (κ/λ) light and a no-template control (NTC) also included per gene. chain restriction [47]. Primers used in validation experiments were identical to those present on the RT Profiler Human Autophagy PCR Array. RNA extraction and cDNA conversion Tissue microarray (TMAs) and IHC analysis Total RNA was isolated from purified single B-cell suspensions or solid LN tissue biopsies with TRIzol® and/ or the RNeasy mini kit. RNA quality was assessed using TMAs were constructed using a semi-automated the Agilent 2100 Bioanalyzer (Agilent Technologies) and arraying system (TMABooster-Alphelys). Sections of a Nanodrop spectrophotometer (Thermo-Scientific); all biopsy material were stained with hemotoxylin and eosin samples had an RNA integrity number (RIN) greater than and reviewed by an expert histopathologist (C.M.) who 6 and 260/280 ratios higher than 1.9. RNA (300ng) was identified areas rich in malignant cells. Triplicate 1mm converted to cDNA by RT-PCR using the RT First Strand cores were then taken from these areas, arrayed and Kit. stained as previously described [31, 33, 34]. Slides were digitalized and image analysis was performed using a digital pathology system (Ariol, Lieca Microsystems). RT2 Profiler PCR array for detection of The Ariol image analysis classifier was trained based on expression of autophagy related genes the hue, saturation and intensity of DAB staining, such that only areas stained above a pre-determined threshold The RT Profiler PCR Array combines qRT- representing the highest intensity staining were classed PCR technology with a microarray format to allow the as positive, and lower level background expression was simultaneous detection of multiple gene expression levels excluded. A further classifier was used to determine the in a rapid manner. The RT Profiler Human Autophagy total viable tissue area. Combining these two classifiers, PCR Array contains primers against 84 genes involved in the percent stained area was calculated. In FL cores, different stages of the autophagy pathway (Suppl Table intra-follicular areas were selected based on expression 6 and 7) as well as primers against five housekeeping of CD10 and clear follicle morphology. Results were genes which are used for data normalization. According manually and blindly reviewed and reported as an average to the manufacturer’s protocol, qRT-PCR was performed of the triplicate cores. Protein levels were expressed as % by adding 2.8ng cDNA mixed with RT SYBR Green stained area [33]. Mastermix (Sabiosiciences) to each well which already contained primers directed against the gene of interest. Western blotting mRNA levels were analyzed using the ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems) and a Proteins were extracted with lysis buffer and 50 µg dissociation curve analysis step included to verify PCR of proteins added to each lane of 4-12% NuPAGE gels and specificity. Relative quantity (RQ) values were calculated -∆∆C Western blotting was performed as previously described using the formula RQ = 2 . Target cycle threshold (C ) T T [33]. values were normalized to the housekeeping gene RPLPO generating a ∆C value. The average ∆C of reactive T T controls was used as the calibrator sample on a per gene Statistical analysis basis and was subtracted from each ∆C generating a delta ∆C value which was linearized by raising to the power of Statistical analysis was performed using GraphPad -∆∆C 2 (2 ). Genes with a fold change ≥3 or ≤-3 or a FC ≥2 Prism software (version 5.03). Data are shown as either or ≤-2 and a p value <0.05 using a Mann-Whitney U test mean ± SD or median with interquartile range when or student t-test were taken to be biologically meaningful. variation was high. Significant differences between groups Hierarchical clustering was performed using Euclidean with unequal size were analyzed with the Mann-Whitney distance measure and an average agglomeration available U test and those with equal size were analyzed using the within the R statistical computing environment. student t-test. Pearson product-moment correlation method was used to analyze linear correlation between two groups. qRT-PCR validation All P-values less than 0.05 were considered statistically significant. Following conversion of RNA (500ng) to cDNA, samples were prepared for qRT-PCR as previously described and the qRT-PCR assay run under the previously www.impactjournals.com/oncotarget 11665 Oncotarget therapy. Oncogene. 2012; 31(8):939-953. Conflict of interest 6. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C, Dipaola The authors declare that they have no conflict of RS, Karantza-Wadsworth V and White E. Autophagy interest. suppresses tumorigenesis through elimination of p62. Cell. 2009; 137(6):1062-1075. Editorial note 7. Jia L, Gopinathan G, Sukumar JT and Gribben JG. Blocking Autophagy Prevents Bortezomib-Induced NF-kappaB This paper has been accepted based in part on peer- Activation by Reducing I-kappaBalpha Degradation in review conducted by another journal and the authors’ Lymphoma Cells. PLoS One. 2012; 7(2):e32584. response and revisions as well as expedited peer-review 8. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson in Oncotarget. D, Chen G, Mukherjee C, Shi Y, Gelinas C, Fan Y, Nelson DA, Jin S and White E. Autophagy promotes tumor Authors’ contribution cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006; 10(1):51-64. 9. Yang ZJ, Chee CE, Huang S and Sinicrope FA. The role of L.J and J.G.G contributed project design and autophagy in cancer: therapeutic implications. Mol Cancer manuscript writing. A.M, A.C, and R.C performed Ther. 2011; 10(9):1533-1541. experiments. A.M, L.J, J.M, and R.D.P contributed data collection and analysis. J.M and A.W provided 10. Vogl DT, Stadtmauer EA, Tan KS, Heitjan DF, Davis LE, clinical information of CLL patients. M.C, R.C and A.C Pontiggia L, Rangwala R, Piao S, Chang YC, Scott EC, contributed histopathological identification of lymphoma Paul TM, Nichols CW, Porter DL, Kaplan J, Mallon G, samples. S.I provided excellent tissue bank service. All Bradner JE, et al. Combined autophagy and proteasome authors contributed manuscript writing. inhibition: A phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma. Autophagy. 2014; 10(8):1380-1390. ACKNOWLEDGEMENTS 11. Lamoureux F, Thomas C, Crafter C, Kumano M, Zhang This project is funded by CRUK PhD studentship to F, Davies BR, Gleave ME and Zoubeidi A. Blocked L.J for supporting A.M. and a program grant P01 CA81538 autophagy using lysosomotropic agents sensitizes resistant from the National Cancer Institute to the CLL Research prostate tumor cells to the novel Akt inhibitor AZD5363. Consortium to JGG. 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