Study of the roles of autophagy in human follicular and diffuse large B-cell lymphoma
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Autophagy, a cellular self-degradation process, plays important roles in cancer development and progression. Autophagy can be inhibited by the anti-apoptotic protein BCL-2 which binds and sequesters the autophagy essential protein Beclin-1, therefore preventing autophagy induction. It is currently unclear whether BCL-2 inhibits autophagy as well as apoptosis in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) which frequently express BCL-2 at high levels. This study aimed to determine (1) the role of BCL-2 in the basal level autophagy status and autophagy flux in primary FL and DLBCL samples, and lymphoma cell lines at both the gene and protein levels; (2) whether aberrant autophagy activity in these lymphoma patients is associated with clinical outcome. We initially found that the BCL-2 inhibitor ABT-737 concurrently induced autophagy and apoptosis in BCL-2HIGH DLBCL cell lines. Blocking autophagy degradation with chloroquine sensitised BCL-2HIGH cells to ABT-737-induced cell death, indicating that acquired autophagy acts as a cytoprotective mechanism in these cells. Expression levels of autophagy-related genes were analyzed by qRT-PCR. The BCL-2HIGH cell line Su-DHL4 showed up-regulation of more autophagy machinery genes at both the basal level and following starvation-induced autophagy compared with the BCL-2LOW cell line. These results suggest that inhibition of BCL-2 can induce cytoprotective autophagy, but overexpression of BCL-2 alone may increase basal level autophagy by inhibiting apoptosis. The expression levels of autophagy-related genes were examined in purified primary FL and DLBCL B cells or un-purified whole FL and DLBCL tumour tissue biopsies and compared with non-malignant reactive lymph nodes. A number of autophagy machinery genes were significantly up-regulated in malignant samples. In particular, more autophagy machinery genes showed significantly increased expression in both purified and un-purified FL samples, indicating that despite frequently overexpressing BCL-2, FL appears to have increased basal level autophagy activity. 4 Expression of the key autophagy proteins p62, Beclin-1, LC3 and BCL-2 were determined in FL and DLBCL using tissue microarrays and immunohistochemistry. Both p62 and LC3, substrates of autophagic degradation, served as markers for autophagy activity. Significantly decreased expression of p62 and LC3, indicating active autophagy, was observed in FL samples (n=117). DLBCL samples (n=109) showed a heterogeneous expression pattern of these four proteins. We identified p62 as an independent prognostic biomarker in DLBCL with decreased expression predicting shorter overall, disease specific and progression-free survival. DLBCL patients with lower p62 or LC3 expression and higher levels of BCL-2, i.e. active autophagy and inhibited apoptosis, had the worst prognosis. Beclin-1 expression was significantly reduced in both FL and DLBCL, where lower levels were significantly associated with shorter overall and disease-specific survival. In summary, this study demonstrates that FL, characterised by overexpression of BCL-2, shows increased autophagy activity, indicating that BCL-2 may not inhibit basal level autophagy in this indolent lymphoma. High levels of BCL-2 and active autophagy did not affect the clinical outcome of FL, but significantly shortened the survival rates of DLBCL patients. Our data propose that active autophagy could be used as a biomarker for DLBCL prognosis, but not FL.
AuthorsMcCarthy, Áine Claire
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