A novel role of HLA class I in the pathology of medulloblastoma
© Smith et al; licensee BioMed Central Ltd. 2009
Received: 19 March 2009
Accepted: 12 July 2009
Published: 12 July 2009
MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma.
We investigated expression of four essential components of MHC class I (heavy chain, β2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro.
The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of β2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells.
MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or β2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.
The host immune system can be harnessed for the treatment of tumors because of the ability of T lymphocytes to specifically recognize tumor-associated antigens. CD8+ T cells destroy tumor cells by perforin-dependent cytotoxic action, following recognition of MHC class I (HLA in humans) molecules at the cell surface . Structurally, the MHC class I molecule is comprised of a 44-kD heavy chain, β2-microglobulin (β2m), and a peptide of 8–10 amino acid residues [2–4]. Presentation of antigenic peptides by MHC class I requires processing involving proteasome-mediated peptide generation from cytosolic proteins, peptide transport into the ER mediated by transporter associated with antigen processing (TAP), and peptide assembly with the heavy chain/β2m heterodimer [3, 4]. TAP is a member of the ATP-binding cassette (ABC)-family of transporters and consists of the TAP1 and TAP2 subunits [3, 5, 6]. Although additional molecules are involved in assembly of MHC class I molecules, the heavy chain, β2m, TAP1 and TAP2 are especially important for functional expression of MHC class I and peptide presentation as deletion of any one of these four molecules results in a profound loss of cell surface expression [3, 4].
Loss of MHC class I by tumor cells is thought to represent evasion of tumors from recognition by tumor-specific CD8+ T cells [7, 8]. Consistent with this notion, loss of MHC class I in cancers such as small cell lung carcinoma, pancreatic carcinoma, cervical cancer, colon cancer and melanoma is a negative prognostic factor [9–12]. In a carefully designed study, HLA class I expression was shown to dramatically decrease in secondary, relative to the primary lesions of breast carcinoma . In another example involving glioma, loss of HLA expression was observed preferentially along the periphery of tumor samples, suggesting that this represents a stealth strategy that facilitates more aggressive tumor expansion . However, a completely opposite correlation was observed in non-small cell lung cancer, uveal melanoma and breast carcinoma, where increased survival occurred in MHC class I negative tumors [15–17]. Interestingly, both positive (HLA-loss) and negative (HLA-down modulation) associations with the outcome of colorectal cancer have been observed in the same study . The reasons for this counter-intuitive effect of MHC class I expression remain elusive.
Besides intercellular interactions, MHC class I molecules have also been implicated in cis modification of signal transduction. Heavy chains dissociated from β2m and peptides (called open conformers), and not the fully assembled MHC class I molecules, associate with a number of cell surface receptors, resulting in modulation of their activation . Interestingly, addition of β2m, which reduces the proportion of open conformers, appears to either reduce  or enhance [21–24] signal transduction in different experimental systems. Thus, the direction of signaling modulation by MHC class I open conformers most likely depends on the identity of the associated receptor and can be manipulated by exogenous addition of β2m. Increased serum levels of β2m were found in several types of cancer [25–32] and were established as a poor prognostic factor in bronchial carcinoma , Hodgkin's lymphoma , multiple myeloma , renal cell carcinoma  and prostate carcinoma . At present, it is unclear whether the effects of β2m are mediated through HLA class I.
Medulloblastoma is the most common pediatric central nervous system malignancy accounting for 30% of all pediatric brain tumors, with the highest prevalence between the ages of three and eight years [33, 34]. Medulloblastoma has the tendency to disseminate throughout the central nervous system early in the course of the disease. The levels of β2m mRNA are significantly higher in metastatic than in non-metastatic forms of medulloblastoma , suggesting a potential association of β2m and/or HLA class I with aggressive behavior and poor prognosis in this type of tumor, as well. Invasiveness in medulloblastoma is mediated by several receptor activation pathways, such as platelet derived growth factor receptor and epidermal growth factor receptor . Common components of these, as well as some other signaling pathways are ERK1/2 and AKT kinases. We show in this manuscript that a small fraction of medulloblastomas express high levels of MHC class I and that markers of poor prognosis are represented disproportionately more in specimens with high MHC class I expression. Furthermore, HLA class I may be involved in signaling modification as exogenously added β2m binds to HLA class I and enhances activation of ERK1/2 that, in turn, affects the mobility of medulloblastoma cells.
Generation of Tissue Microarray
Medulloblastoma tissue microarrays were generated as previously described . Formalin-fixed, paraffin embedded tissue blocks were obtained from both the Armed Forces Institute of Pathology and Children's National Medical Center. More than 95% of tissues were obtained for diagnostic purposes, hence patients underwent no prior therapy. A neuropathologist (MS) marked two representative tumor core regions within each tissue block. The cores measuring 0.6 mm in diameter and 3–4 mm in height were isolated. A de-identified microarray was generated using 4–8 μm sections cut from the core blocks including: 139 medulloblastomas (47 classic, 40 anaplastic, 25 desmoplastic and 27 with unclassified histology), 21 primitive neuroectodermal tumors, 10 small cell carcinomas, 5 atypical teratoid rhabdoid tumors, 3 oat cell lung carcinomas, one each of ependymoblastoma and lymphoma, and 20 tissues of brain metastases from tumors of various origin. IRB approval was obtained for the construction and analysis of these tissue microarrays and all other tumor specimens investigated.
DAOY, D283 and D556 medulloblastoma cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2 in RPMI media supplemented with 10% Fetal bovine serum, 2 mM L-glutamine, 1 mM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin.
Polyclonal rabbit anti-human β2m and monoclonal mouse anti-human CD45 (clones 2B11 + PD7/26) were purchased from DakoCytomation, Carpinteria, CA. The hybridoma secreting HC-10 antibody, specific for HLA heavy chain epitope revealed in the absence of β2m and peptide , was kindly provided by Dr. Pan Zheng (University of Michigan School of Medicine, Ann Arbor, MI). Anti- TAP1 (NOB-1) and TAP2 (NOB-2) antibodies  were provided by Dr. S. Ferrone (Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA). The W6/32 monoclonal antibody is specific for HLA A, B and C and specifically to the combined epitope contributed by the α2 and α3 domains of the heavy chain and β2m [40–42]. mAb rabbit anti-human GAPDH, rabbit anti-human phospho-p44/p42 MAP kinase (Thr202/Tyr204), mAb rabbit anti-human p44/p42 MAP kinase, rabbit anti-human phospho-AKT (Ser473), rabbit anti-human AKT, mAb mouse anti-human phospho-EGF receptor (Tyr1068), rabbit anti-human EGF receptor, rabbit anti-human phospho-PDGFRβ (Tyr751) were purchased from Cell Signaling (Beverly, MA), anti-c-myc from Abcam (Cambridge, MA), rabbit anti-human PDGFR- β (Santa Cruz) and anti-rabbit IgG HRP-linked antibody (Cell Signaling). HC-10 and W6/32 antibodies were partially purified from hybridoma supernatants using the centriplus YM-100 (Millipore) regenerated cellulose filters that have a molecular weight cutoff of 100,000 Da. Antibody concentration after purification was 2 mg/ml.
Slides containing tissue arrays were deparaffinized and rehydrated. The primary Abs HC-10, TAP1 and TAP2 required an additional heat-induced epitope retrieval step (Target Retrieval Solution pH 9 or Target Retrieval Solution, pH6.1, DakoCytomation, Carpinteria, CA). Slides were blocked with biotin (Biotin Blocking System, DakoCytomation) and processed using the 3-step streptavidin-biotin-immunoperoxidase staining system (LSABTM2+ System-HRP, DakoCytomation, Carpinteria, CA). Diaminobenzidine (DAB) was the chromogenic substrate. Mayers hematoxylin was used as a counterstain and followed with an ammonia wash. Slides were mounted using an aqueous mounting medium (Faramount, DakoCytomation). Controls consisted of parallel sections without primary antibody. Stainings for heavy chain, β2m, TAP1, TAP2 and CD45 were assigned scores of 0–3  based on the percentage of stained cells (0- no staining; 1- less than 10% cells stained; 2- 10–50% cells stained; 3- >50% cells positive). The staining for c-myc was not restricted to particular cells, but was rather present or absent throughout the tumor. Hence, c-myc scores were assigned 0 (absent staining), 1 (weak diffuse staining) or 2 (strong diffuse staining). Each score was an average of the two samples graded for each individual tumor in the array. Each slide was evaluated by three independent graders including two neuropathologists. The images were acquired using the following equipment: Microscope – Carl Zeiss Axioskop; Lenses- Achroplan 40× and Achroplan 20×; Camera – Carl Zeiss Axio Cam HRC; Acquisition software- Axio Vision Rel. 4.3.
Cells were incubated with either 5% FBS in PBS (controls) or W6/32 or HC-10 supernatant for 1 hour. After 3 rinses, cells were incubated with PE labeled donkey anti-mouse IgG (H+L) (eBiosciences, San Diego, CA) for 30 minutes at 4°C. Cells were rinsed 3 times and fixed in cytofix (BD Sciences, San Diego, CA).
Total cellular RNA, obtained from 10 frozen medulloblastoma specimens, was subjected to the SuperScript First-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) to generate the cDNA for the PCR reactions. RT-PCR were performed using 35 cycles of 30 seconds at 94°C, 1 minute at the annealing temperature, and 1 minute at 72°C (with the exception for HLA-A and HLA-B where the last cycle was for 1 minute 30 seconds at 72°C). The annealing temperatures were 57°C for TAP2 and β2m, 58°C for β-actin and CD45, 59°C for TAP1 and 60°C for HLA-A and HLA-B. The primer sequences were: 5'-GAGACATCTTGGAACTGGAC-3' and 5'-CTCTGAGTGAGAATCTGAGC-3' (forward and reverse, TAP1), 5'-GTACAACACCCGCCATCAG-3' and 5'-GGACGTAGGGTAAACGTCAGC-3' (TAP2), 5'-CTCGCGCTACTCTCTCTT-3' and 5'-AAGACCAGTCCTTGCTGA-3' (β2m), 5'-TATAGTCGACCACCCGGACTCAGAATCTCCT-3' and 5'-ATATGGATCCATCTCAGTCCCTCACAAGA-3' (HLA-A), 5'-TATAGTCGACCACCCGGACTCAGAGTCTCCT-3' and 5'-ATATGGATCCATCTCAGTCCCTCACAAGA-3' (HLA-B), 5'-ACCTGTACGCCAACACAGTG-3' and 5'-GCCATGCCAATCTCATCTT-3' (β-Actin), 5'-CTGAAGGAGACCATTGGTGA and 5'-GGTACTGGTACACAGTTCGA-3' (CD45). The reactions were loaded onto a 1% agarose gel and products were visualized by ethidium bromide staining.
DAOY and D283 cells were cultured in serum-free media for 24 hrs at 37°C. Cells were washed twice and treated with serum-free RPMI with or without human β2m (Lee Biosolutions, Inc, St. Louis, Missouri) or monoclonal antibodies for indicated times, at 37°C. Signaling was halted by the addition of ice cold PBS and cells were lysed with 1× cell lysis buffer (Cell Signaling, Danvers, Massachussetts) supplemented with PhosSTOP phosphatase inhibitor cocktail and complete mini protease inhibitor cocktail (Roche, Indianapolis, Indiana). Lysates were centrifuged at 14,000 rpm for 15 minutes to remove cell debris, boiled for 5 minutes in loading buffer and separated by a 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, California). Membranes were blocked in 5% milk in TBST for 1 hour at room temperature. Primary and secondary antibodies were diluted in 3% BSA in TBST and incubated with the membrane at 4°C overnight and at room temperature for 1 hour, respectively. Signal was detected using the enhanced chemiluminescence system (Pierce, Rockford, IL). Densitometry quantification of the bands was performed using Quantity One software (Bio-Rad, Hercules, CA), according to manufacturer's instructions. The Band 1/Band 2 ratio was obtained by dividing differences between intensity units observed in the square areas containing specific bands and an identical blank area drawn in the immediate vicinity of the band, according to the following formula: Band 1/Band 2 ratio= (Band 1-blank)/(Band 2 -blank).
Wound scratch assay
Wound scratch assay was used to evaluate tumor cell migration [44, 45]. DAOY cells were plated in a 60 mm dish at 40% confluency and grown overnight. After 24 hours the cells were scraped down the middle of the plate with a 200 μl pipette tip to induce the resemblance of a wound and washed twice with serum-free media. Cells were cultured with serum free media with or without 2 μg/ml β2m for 24 hours. Images of the wound were taken at 0 and 24 hours. The area of the wound was calculated using the Axiovision system by tracing the cleared space. Area was measured in μM2. The ratio of 24 hours to 0 hours was calculated for each sample.
Medulloblastoma expression of MHC class I antigen
Summary of HLA class I and CD45 staining scores for medulloblastoma array.
To determine whether expression of the components of MHC class I processing machinery could be ascribed to infiltrating leukocytes, we stained the medulloblastoma microarray with anti-CD45 antibody (Fig. 1C). Of the 103 evaluable medulloblastoma specimens, 58.3% were negative for infiltration with 39.8% receiving a score of 1 and 0.95% a score of 2 and 3 each (Table 1). The positive staining samples showed leukocytes present in the tumor, but few that had migrated away from the blood vessels into the tissue. The low levels of infiltration appear to be indicative of the expected poor immune response associated with medulloblastomas. Of the 14 MHC class I-high samples, 6 were negative for leukocyte infiltration and 6 received a score of 1. Scores of 2 and 3 were noted in one sample each. Therefore, contamination by infiltrating peripheral white blood cells cannot account for positive heavy chain, TAP1, TAP2 and β2m staining in most of these cases and MHC class I expression appears to be genuinely derived from medulloblastoma cells.
Association of MHC class I expression and anaplastic medulloblastoma subtype
Anaplastic histopathology [48, 49] and c-myc expression [49–51] are negative prognostic markers for medulloblastoma. To test whether MHC class I expression had any prognostic value for medulloblastomas, we evaluated the distribution of histological subtypes and expression of c-myc in specimens with scores 2 or higher for heavy chain versus those that scored less than 2. The reason for the cut-off at score 2 is that score 1 was given if only up to 10% cells in the specimen stained positive. In contrast to the diffuse character of the anaplasia and c-myc expression, we considered that the impact, if any, of so few positive cells on the overall histology of the tumor could not have been significant.
Binding of exogenous β2m alters the balance of open and closed MHC class I conformers in medulloblastoma cell line
Engagement of open MHC class I conformers modulates phosphorylation of ERK1/2
Increased migration of DAOY cells in the presence of β2m
We show herein that classical MHC class I is undetectable in the majority of medulloblastomas. HLA expression by medulloblastoma was reported twice previously. Bodey et al. analyzed the infiltrating cell phenotype in 34 medulloblastomas and 42 astrocytomas . The authors noted that neoplastically transformed cells expressed HLA-A, -B, and -C molecules within all 76 tissues. Since this study focused on the phenotype of infiltrating cells little detail was provided about HLA expression by tumor cells. In another study of 10 medulloblastomas, none of the tumors showed HLA expression . One major advantage of this study is that MHC class I processing machinery was analyzed in greater detail, by examining expression of HLA heavy and light chains and TAP2 subunit. In addition, other molecules, such as LMP2 and LMP7 that are involved, but not essential for MHC class I processing, were studied. The present investigation combined elements from both previous ones, including a large patient sample size and analysis of expression of several molecules required for HLA class I assembly. The large sample size enabled us to detect a minority of medulloblastomas that express HLA class I, that may be easily missed in smaller sample collections. Importantly, we were able to exclude infiltrating leukocytes as a source of HLA class I expression and analyze it in relation to the histological subtypes of medulloblastoma.
MHC class I- negative tumor cells may arise due to multiple mutations in genetically unstable cancer cells and subsequent selection by tumor-specific CD8+ T cells [7, 8]. The negative prognostic impact of a loss of MHC class I in small cell lung carcinoma, pancreatic carcinoma, cervical cancer, colon cancer and melanoma is consistent with this view [9–12]. Alternatively, HLA-negative may have arisen by transformation of cells that originally did not express MHC class I. These examples would include well-differentiated cells, such as muscle cells and neurons, unless the expression is induced by inflammatory stimuli. Because medulloblastoma develops from neuronal precursors and the CNS is relatively protected from the immune system, we consider selection by CD8+ T cells an unlikely cause of the HLA-negative phenotype of most medulloblastomas. An explanation for HLA class I expression by a minority of medulloblastomas remains to be established.
The expression of c-myc and/or presence of anaplasia, which are negative prognostic markers for medulloblastoma [48–51], were associated with HLA class I expression. Thus, HLA class I expression may be associated with more aggressive medulloblastomas. If the association is confirmed in larger studies, MHC class I expression may prove to be an important biomarker of the malignant phenotype. Because follow-up clinical data were not available for all patients in this study, we were unable to make correlation between high HLA class I expression and disease outcomes. Nevertheless, the association of higher expression of β2m with metastatic disease in a previous study of ours  is consistent with the findings of the present study. Despite considerable evidence suggesting that the loss of MHC class I by tumor cells may indicate escape from immune surveillance, there are recognized examples, including non-small cell lung cancer, uveal melanoma and breast carcinoma, where increased survival correlates with downregulation of MHC class I [15–17]. This paradoxical effect may be mediated by the activity of NK cells, a cell type that is generally more effective when target cells do not display MHC class I . Alternatively, β2m  and heavy chain devoid of peptide and β2m , may be implicated in modifying signal transduction, suggesting an immune system-independent role of MHC class I subunits in tumor progression. Indeed, the latter possibility was demonstrated by the ability of β2m to modulate the phosphorylation of ERK1/2. Binding of the exogenous β2m to open forms of MHC class I was likely responsible for this effect because antibody specific for the HLA open forms inhibited the binding and could mimic the signal modulation effect.
Dysregulation of ERK1/2 has been implicated in tumorigenesis of various cell types. Mitogen-activated protein kinases are involved in the cellular response to stimuli resulting in activation of membrane, cytoplasmic and nuclear signaling pathways. ERK1/2 has a role in the phosphorylation of cytoplasmic and nuclear targets, regulation of cell proliferation, differentiation, survival, angiogenesis, migration and chromatin remodeling [62, 63]. Our results show a role of ERK1/2 activation in promoting migration of medulloblastoma cells in vitro. This may explain previously found higher levels of β2m expression in metastatic medulloblastoma . Excess of β2m protein produced and secreted into the extracellular matrix of medulloblastoma tissues could bind to HLA class I open conformers on neighboring cells, enhancing activation of ERK1/2 and the invasive capability of medulloblastoma cells.
In summary, we found a novel role of MHC class I contributing towards a more malignant phenotype of medulloblastoma by enhancing the activation of ERK1/2. The contrasting effects of MHC class I on different tumors could be explained by differential effects of MHC class I on individual cell surface receptor signaling and/or by the presence or absence of an overriding tumor-specific CD8+ T cell response.
transporter associated with antigen processing.
Courtney Smith was a predoctoral student in the Immunology Program of the Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation that was presented to the above program in partial fulfillment of the requirements for the Ph.D. degree. The authors thank Drs. Pan Zheng and Soldano Ferrone for providing HC-10 and TAP-specific antibodies, respectively, Mary Rose for the gift of primers for β-actin and Thamara Abouantoun for the help with Western blotting. These studies were supported in part by an R01 grant # CA111835 from NIH to TJM.
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