Beclin 1, Lc3 Expression In Papillary Thyroid Carcinoma And Breast Carcinoma: Correlation With Lymph Node Metastasis

BackgroundAutophagy is an evolutionarily conserved catabolic process of self-digestion whereby double-membrane vesicles engulfing cellular organelles and cytoplasm form in the cytosol and fuse with lysosomes, where the sequestered contents are degraded and recycled for protein and ATP synthesis. It is an evolutionarily conserved process that occurs in all eukaryotic cells, from yeast to mammals. At the beginning of autophagy, portions of the cytoplasm, as well as intracellular organelles, are sequestered in double-membranebound structures that are known as autophagoaosmes. These autophagosomes then fuse with lysosomes to form autolysosomes, and the sequestered contents are degraded by lysosomal hydrolases and are recycled. In addition to its basic role in the turnover of proteins and organelles, autophagy has multiple physiological and pathophysiological functions. When cells encounter environmental stressors such as nutrient starvation and pathogen infection, autophagy takes place, resulting in either adaptation and survival, or death. There is accumulating evidence that autophagy is believed to have an important role in tumour development and metastasis. Autophagy is involved in both the recycling of normal cellular constituents and the removal of damaged proteins and organelles. The early stages of tumour development require cancer cells to undergo a higher level of protein synthesis than protein degradation for the tumour to grow. As autophagy can provide essential nutrients necessary for cells to survive, it is conceivable that autophagy may be used to provide nutrients to other cells in multicellular organism.Beclinl is the mammalian orthologue of the essential yeast ATG6/VPS30 gene, which is required for autophagosome formation in a complex with the class III phosphatidylinositol-3-kinase (PIK3C3 or Vps34). Beclinl complements the autophagy defect present in atg6/vps30-disrupted yeast and in human MCF7 breast cancer cells. The human Beclinl gene is on chromosome 17q21 within a region commonly allelically deleted in ovarian, breast and prostate cancers. Many breast carcinoma cell lines, although polyploid for chromosome 17, exhibit deletions of one or more Beclinl alleles and human breast tumors show decreased Beclinl levels compared to normal adjacent tissue. Restoration of Beclinl and autophagy in MCF-7 cells is associated with inhibition of MCF7-induced tumorigenesis in nude mice. Beclinl-/-mice die early in embryogenesis, likely due to loss of the essential role of autophagy during development. Mammary tissue from aging Beclinl+/-mice shows hyperproliferative, preneoplastic changes, indicative of a correlation between low Beclinl levels and mammary tumorigenesis.In mammalian autophagy, LC3 protein, a mammalian homolog of the yeast Atg8p, is used as an index of autophagosome formation. Following the synthesis as Pro-LC3, C-terminal fragment is cleaved off by hAtg4Bp to yield a cytosolic precursor form, LC3-I (soluble, unlipidated). LC3-I subsequently conjugates with a phospholipid via an ubiquitylation-like system through hAtg7p and hAtg3p, to form the active LC3-Ⅱ(lipidated, membrane bounded), which is then targeted to the early autophagosome membrane.LC3-Ⅱis localized to pre-autophagosomes and autophagosomes, making this protein an autophagosomal marker. Following the fusion of autophagosomes with lysosomes, intra autophagosomal LC3-Ⅱis rapidly degraded by lysosomal hydrolytic enzymes. Introduction of autophagy by various stresses such as starvation stimulates the conversion of LC3-Ⅰto LC3-Ⅱ, and upregulation of LC3 expression. Thus, LC3 is a specific marker of autophagosome formation.Thyroid carcinoma represents 90% of all endocrine malignancies, with papillary thyroid cancer(PTC) accounting for 80% of all thyroid cancers. The lymph node involvement in papillary thyroid carcinoma is commonly detected, reportedly up to 70%. CADY and coworkers reported recurrence and death rates of 33% and 27% for high-risk patients vs 11% and 4% for low-risk patients, respectively. Our recent study revealed that point mutation of the Beclinl gene is a rare event in papillary thyroid carcinoma (PTC). Rather, increased expression of Beclinl in the cancer nest compared to their normal tissue, and also in metastatic lymph node compared to normal lymph node suggest that neo-expression of Beclinl may play a role in tumorigenesis and lymph node metastasis in human PTCObjectiveWe investigate the relationship between Beclinl, Lc3 protein expression in papillary thyroid cancer tissues and normal thyroid tissues, metastatic lymph nodes and normal lymph nodes.MethodsWe investigated the expression of Beclinl, Lc3 in human PTC. Tissue samples from 86 cases of papillary thyroid carcinoma were used for the present study.57 cases of papillary thyroid carcinoma were with lymph node metastasis. The expression of Beclinl, Lc3 in tumor, normal tissue adjacent to tumor, distant normal tissue, metastatic lymph node and normal lymph node was examined with immunohistochemistry. The Beclinl, Lc3 expression between tumor and normal tissue, metastatic and normal lymph node was also analyzed.ResultsBeclinl, Lc3 is overexpression in primary tumors of PTC patientsIn the papillary thyroid carcinoma analyzed, immunopositivity (defined as≥30% of the tumor cells) was observed for Beclinl in 76 (88.4%) of the 86 tumor samples and Lc3 in 76 (88.4%) of the 86 tumor samples. We also scored the extent of Beclinl-positive carcinoma cells 10 and 66 cases, respectively, were++and+++ Beclinl-positive. Immunostaining of Beclinl, Lc3 was observed on the membrane-plasma, cytoplasm and nucleus in the cancer cells, whereas negative or weak stain of Beclinl was observed in the adjacent noncancerous tissue and diatant normal tissue. When compared, intensity of immunostaining in the thyroid cancer cells was stronger than in the normal cells in 76 (88.4%) thyroid cancer tissues. The immunostaining was judged to be specific by several criteria, including absence of consistent immunostaining of cells with normal rabbit serum at the same dilution and reduction of signal intensity as the dilution of the antibody was increased.Beclin1, Lc3 is overexpression in lymph node metastases of PTC patientsImmunostaining of Beclin1 was observed in 56 (98.2%) of the 57 metastatic lymph nodes and Lc3 was observed in 56 (98.2%) of the 57 metastatic lymph nodes. We also scored the extent of Beclinl positive metastatic carcinoma cells 8 and 48 cases, respectively, were++and+++Beclin1-positive. Immunostaining of Beclin1, Lc3 when present, was cytoplasmic and membrane-plasma. No or weak stain of Beclin1, Lc3 was observed in the normal lymph nodes.Western blot analysisTo confirm the specificity of Beclin1 antibody, a set of freshly harvested PTC specimens, normal tissue adjacent to tumor, distant normal tissue, metastatic lymph node and normal lymph node were subjected to Western blot analysis. As shown in, protein bands immunopositive for Beclinl forms were clearly evident in each sample. Distant normal thyroid tissues generally yielded no, or only a faint, band for Beclin1, Lc3 and normal tissues adjacent to tumor yielded weak band for Beclin1, Lc3. All tumor tissues yielded stronger bands for Beclin1, Lc3 compared with noncancerous thyroid tissues. There were higher levels of Beclin1, Lc3 in tumor tissues than in normal thyroid tissues.Normal lymph nodes generally yielded no, or only a faint, band for Beclin1, Lc3. Metastatic lymph nodes yielded stronger bands for Beclin1, Lc3 compared with normal lymph nodes. There were higher levels of Beclin1, Lc3 in metastatic lymph nodes than in normal lymph nodes.Beclin1, Lc3 expression is correlated with lymph node metastasisThere was no significant correlation of the Beclin1, Lc3 expression with the clinicopathological parameters, such as age, sex, histological differentiation, tumor size, tumor site, tumor extracapsularity, although extracapsular invasion is a high-risk factor of lymph node metastasis (Fisher’s exact test, p>0.05). It was higher in the lymph node metastasis group than in the no lymph node metastasis group (Fisher’s exact test, p=0.018). Logistic regression analysis revealed that the expression of Beclin1, Lc3 was significantly associated with lymph node metastasis (p=0.004).Conclusion Our data indicate that Beclinl, Lc3 inactivation by loss of expression may not occur in PTC. Rather, increased expression of Beclinl, Lc3 in the cancer nest compared to their normal tissue, and also in metastatic lymph node compared to normal lymph node suggest that neo-expression of Beclinl, Lc3 may play a role in tumorigenesis and lymph node metastasis in human PTC.