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Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer.

Chen L, Dahlstrom JE, Chandra A, Board P, Rangasamy D - Breast Cancer Res. Treat. (2012)

Bottom Line: The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %.Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression.Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia.

ABSTRACT
Long interspersed nuclear element 1 (L1) belongs to a family of retrotransposons. Expression of the normally repressed L1 retrotransposons has been shown to induce genome instability by creating DNA double-stranded breaks and chromosomal rearrangements through the process of retrotransposition. At present, little is known about the expression of L1-encoded ORF1p and ORF2p which are indispensable for its retrotransposition activity. Given its potentially harmful effects on the genome, we investigated the implications of both ORF1p and ORF2p expression and their subcellular localization in a range of breast cancer cell lines and breast tumor tissues including 15 normal breast tissues, 25 fibroadenomas, 25 ductal carcinomas in situ (DCIS), and 95 invasive cancers. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of ORF1p and ORF2p using univariate and multivariate analysis. High cytoplasmic expression of ORF1p and ORF2p was seen in DCIS tumors, but they were not related with survival outcome. The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %. Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression. This is the first study examining the effects of both ORF1p and ORF2p expression in breast cancer tissues. Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival. The differing patterns of both cytoplasmic and nuclear ORF1p and ORF2p expression indicate that further studies of the biology and function of L1 retrotransposons are required in breast cancer.

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Aberrant expression of L1 retrotransposons in breast cancer cells. A Whole-cell lysates of breast cancer cell lines were analyzed by western blotting with anti-ORF1p and anti-ORF2p antibodies. As a loading control, α-tubulin was used. Cell lysates from N.Tera.2D1 were used as positive controls. Both ORF1p and ORF2p specifically expressed in cancer cell lines but not in non-tumorigenic HMEC and MCF10A cells. B Cell blocks taken from the normal HMEC, poorly invasive T47D and highly invasive metastatic MDA-MB-231 cell lines were stained immunohistochemically using the anti-ORF1p and anti-ORF2p antibodies. HMEC cells did not stain, but T47D cells displayed strong cytoplasmic staining, whereas MDA-MB-231 cells showed moderate-to-strong nuclear staining. The merged panel of Hematoxylin (nucleus) and ORF1p or ORF2p are shown. Bar represents 20-μm. C The immunofluorescence analysis of ORF1p expression. L1-encoded proteins appear to localize in both the cytoplasm and nucleus of cell lines, although the intensity of staining varies between them. Nuclei were stained with DAPI (blue) and ORF1p (green). NTera.2D1 and HMEC cells were used as positive and negative controls, respectively. D Overexpressions of ORF1p and ORF2p in breast tumors. Protein extracts from breast tumors (T) and their adjacent normal breast tissues (N) were isolated from two different patients and performed western blotting. Using the mixture of both anti-ORF1p and anti-ORF2p antibodies, the expressions of ORF1p and ORF2p were detected only in breast tumors. For protein normalization, α-tubulin was used as a loading control
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Fig2: Aberrant expression of L1 retrotransposons in breast cancer cells. A Whole-cell lysates of breast cancer cell lines were analyzed by western blotting with anti-ORF1p and anti-ORF2p antibodies. As a loading control, α-tubulin was used. Cell lysates from N.Tera.2D1 were used as positive controls. Both ORF1p and ORF2p specifically expressed in cancer cell lines but not in non-tumorigenic HMEC and MCF10A cells. B Cell blocks taken from the normal HMEC, poorly invasive T47D and highly invasive metastatic MDA-MB-231 cell lines were stained immunohistochemically using the anti-ORF1p and anti-ORF2p antibodies. HMEC cells did not stain, but T47D cells displayed strong cytoplasmic staining, whereas MDA-MB-231 cells showed moderate-to-strong nuclear staining. The merged panel of Hematoxylin (nucleus) and ORF1p or ORF2p are shown. Bar represents 20-μm. C The immunofluorescence analysis of ORF1p expression. L1-encoded proteins appear to localize in both the cytoplasm and nucleus of cell lines, although the intensity of staining varies between them. Nuclei were stained with DAPI (blue) and ORF1p (green). NTera.2D1 and HMEC cells were used as positive and negative controls, respectively. D Overexpressions of ORF1p and ORF2p in breast tumors. Protein extracts from breast tumors (T) and their adjacent normal breast tissues (N) were isolated from two different patients and performed western blotting. Using the mixture of both anti-ORF1p and anti-ORF2p antibodies, the expressions of ORF1p and ORF2p were detected only in breast tumors. For protein normalization, α-tubulin was used as a loading control

Mentions: Earlier studies revealed that the level of ORF1p is significantly elevated in breast cancer cell lines [26, 27]. A recent study of clinical samples has also shown that expression of ORF1p is widespread in breast tumors [25], but the expression levels of ORF2p remain unknown. To gain insight into L1 expression, we first determined the ORF1p and ORF2p expression in breast cancer cell lines by Western blot analysis. Clinicopathologic features of these cells are summarized in Table S1. Human embryonic carcinoma NTera.2D1 cells were used as a positive control. As shown in Fig. 2A, both ORF1p and ORF2p were overexpressed in all tested breast cancer cell lysates (T47D, SKBR3, BT-20, MDA-MB-361, MCF-7, Hs578T, MDA-MB-231, and MDA-MB-436), but not in non-tumorigenic breast epithelial HMECs or its derivative MCF10A cell line. Notably, the relative expression levels of ORF1p and ORF2p were markedly higher in low-invasive cancer cells (T47D, SKBR3, and BT20) compared with moderate (MCF7 and Hs578T)-to-highly invasive breast cancer (MDA-MB-231, MDA-MB-436) cell lines. The increased expression of both proteins was further confirmed using immunohistochemistry and immunofluorescence assay (Fig. 2B, C). Interestingly, the relative levels of ORF2p expression were lower in all cell lines compared with ORF1p. While ORF2p shows weak-to-moderate fluorescent staining, ORF1p exhibits high fluorescence signals. This differential expression is consistent with previous reports from Eugun et al. [24] who detected differential levels of ORF2p expression in various fetal and adult tissues. Lower expression of ORF2p compared with ORF1p was also recently reported in vascular endothelial cells [28].Fig. 2


Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer.

Chen L, Dahlstrom JE, Chandra A, Board P, Rangasamy D - Breast Cancer Res. Treat. (2012)

Aberrant expression of L1 retrotransposons in breast cancer cells. A Whole-cell lysates of breast cancer cell lines were analyzed by western blotting with anti-ORF1p and anti-ORF2p antibodies. As a loading control, α-tubulin was used. Cell lysates from N.Tera.2D1 were used as positive controls. Both ORF1p and ORF2p specifically expressed in cancer cell lines but not in non-tumorigenic HMEC and MCF10A cells. B Cell blocks taken from the normal HMEC, poorly invasive T47D and highly invasive metastatic MDA-MB-231 cell lines were stained immunohistochemically using the anti-ORF1p and anti-ORF2p antibodies. HMEC cells did not stain, but T47D cells displayed strong cytoplasmic staining, whereas MDA-MB-231 cells showed moderate-to-strong nuclear staining. The merged panel of Hematoxylin (nucleus) and ORF1p or ORF2p are shown. Bar represents 20-μm. C The immunofluorescence analysis of ORF1p expression. L1-encoded proteins appear to localize in both the cytoplasm and nucleus of cell lines, although the intensity of staining varies between them. Nuclei were stained with DAPI (blue) and ORF1p (green). NTera.2D1 and HMEC cells were used as positive and negative controls, respectively. D Overexpressions of ORF1p and ORF2p in breast tumors. Protein extracts from breast tumors (T) and their adjacent normal breast tissues (N) were isolated from two different patients and performed western blotting. Using the mixture of both anti-ORF1p and anti-ORF2p antibodies, the expressions of ORF1p and ORF2p were detected only in breast tumors. For protein normalization, α-tubulin was used as a loading control
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Related In: Results  -  Collection

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Fig2: Aberrant expression of L1 retrotransposons in breast cancer cells. A Whole-cell lysates of breast cancer cell lines were analyzed by western blotting with anti-ORF1p and anti-ORF2p antibodies. As a loading control, α-tubulin was used. Cell lysates from N.Tera.2D1 were used as positive controls. Both ORF1p and ORF2p specifically expressed in cancer cell lines but not in non-tumorigenic HMEC and MCF10A cells. B Cell blocks taken from the normal HMEC, poorly invasive T47D and highly invasive metastatic MDA-MB-231 cell lines were stained immunohistochemically using the anti-ORF1p and anti-ORF2p antibodies. HMEC cells did not stain, but T47D cells displayed strong cytoplasmic staining, whereas MDA-MB-231 cells showed moderate-to-strong nuclear staining. The merged panel of Hematoxylin (nucleus) and ORF1p or ORF2p are shown. Bar represents 20-μm. C The immunofluorescence analysis of ORF1p expression. L1-encoded proteins appear to localize in both the cytoplasm and nucleus of cell lines, although the intensity of staining varies between them. Nuclei were stained with DAPI (blue) and ORF1p (green). NTera.2D1 and HMEC cells were used as positive and negative controls, respectively. D Overexpressions of ORF1p and ORF2p in breast tumors. Protein extracts from breast tumors (T) and their adjacent normal breast tissues (N) were isolated from two different patients and performed western blotting. Using the mixture of both anti-ORF1p and anti-ORF2p antibodies, the expressions of ORF1p and ORF2p were detected only in breast tumors. For protein normalization, α-tubulin was used as a loading control
Mentions: Earlier studies revealed that the level of ORF1p is significantly elevated in breast cancer cell lines [26, 27]. A recent study of clinical samples has also shown that expression of ORF1p is widespread in breast tumors [25], but the expression levels of ORF2p remain unknown. To gain insight into L1 expression, we first determined the ORF1p and ORF2p expression in breast cancer cell lines by Western blot analysis. Clinicopathologic features of these cells are summarized in Table S1. Human embryonic carcinoma NTera.2D1 cells were used as a positive control. As shown in Fig. 2A, both ORF1p and ORF2p were overexpressed in all tested breast cancer cell lysates (T47D, SKBR3, BT-20, MDA-MB-361, MCF-7, Hs578T, MDA-MB-231, and MDA-MB-436), but not in non-tumorigenic breast epithelial HMECs or its derivative MCF10A cell line. Notably, the relative expression levels of ORF1p and ORF2p were markedly higher in low-invasive cancer cells (T47D, SKBR3, and BT20) compared with moderate (MCF7 and Hs578T)-to-highly invasive breast cancer (MDA-MB-231, MDA-MB-436) cell lines. The increased expression of both proteins was further confirmed using immunohistochemistry and immunofluorescence assay (Fig. 2B, C). Interestingly, the relative levels of ORF2p expression were lower in all cell lines compared with ORF1p. While ORF2p shows weak-to-moderate fluorescent staining, ORF1p exhibits high fluorescence signals. This differential expression is consistent with previous reports from Eugun et al. [24] who detected differential levels of ORF2p expression in various fetal and adult tissues. Lower expression of ORF2p compared with ORF1p was also recently reported in vascular endothelial cells [28].Fig. 2

Bottom Line: The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %.Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression.Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia.

ABSTRACT
Long interspersed nuclear element 1 (L1) belongs to a family of retrotransposons. Expression of the normally repressed L1 retrotransposons has been shown to induce genome instability by creating DNA double-stranded breaks and chromosomal rearrangements through the process of retrotransposition. At present, little is known about the expression of L1-encoded ORF1p and ORF2p which are indispensable for its retrotransposition activity. Given its potentially harmful effects on the genome, we investigated the implications of both ORF1p and ORF2p expression and their subcellular localization in a range of breast cancer cell lines and breast tumor tissues including 15 normal breast tissues, 25 fibroadenomas, 25 ductal carcinomas in situ (DCIS), and 95 invasive cancers. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of ORF1p and ORF2p using univariate and multivariate analysis. High cytoplasmic expression of ORF1p and ORF2p was seen in DCIS tumors, but they were not related with survival outcome. The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %. Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression. This is the first study examining the effects of both ORF1p and ORF2p expression in breast cancer tissues. Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival. The differing patterns of both cytoplasmic and nuclear ORF1p and ORF2p expression indicate that further studies of the biology and function of L1 retrotransposons are required in breast cancer.

Show MeSH
Related in: MedlinePlus