Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation

Abstract

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues.We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively.Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker.

Keywords: LINE-1/L1; ORF2; retrotransposon; reverse transcriptase; tumorigenesis.

Figure 1. chA1-L1 monoclonal antibody specifically recognizes both endogenous and overexpressed ORF2p in A-375 melanoma cells

(A) Peptide competition assay. Whole cell extract (50 μg/lane) from A-375 melanoma cells were stained with Ponceau S (lanes 1 and 2); filters were incubated with chA1-L1 mAb pre-incubated with peptide 39 (lane 3) or mAb alone (lane 4). chA1-L1 mAb identifies a single band at the predicted migration for ORF2p molecular mass, 150 kDa; peptide 39 abrogates the binding of chA1-L1 with the antigen; α-tubulin served as loading control. (B) Immunoblot analysis of whole cell extract (50 μg) from A-375 cells interfered with vector (pS-neo, control), or with L1-interfering shRNAs (pS-L1i), using chA1-L1 antibody; α-tubulin is used as a loading control. Histograms represent the densitometric quantification of band signal intensities; data are shown as fold change relative to control (pS-neo) after normalization to α-tubulin. Data are expressed as mean ± S.D. of three independent experiments; *P < 0.05 (paired t test). (C) Immunoblot analysis of cell extract from pTT5-L1-, mock- and pCMV-hGH-transfected A-375 cells using chA1-L1 mAb to detect transiently overexpressed ORF2p. Notably, 5 μg of cell extract (i.e., 10-fold less than in A and B) were loaded on the gel; α-tubulin served as loading control. (D) Immunofluorescence assay of mock- (a), pTT5-L1- (b) and pCMV-hGH-transfected (c) A-375 cells. Cells were stained with chA1-L1 mAb and DAPI to detect ORF2p (green) and cell nuclei (blue), respectively. chA1-L1 mAb is omitted in the negative control (d). Bar, 10 μm; magnification 100x. Higher magnification of the boxed areas in (a′), (b′) and (c′).

Figure 2. Immunofluorescence assay of A-375 cells stained with anti-ORF1p polyclonal (red, a) and chA1-L1 anti-ORF2p monoclonal (green, b) antibodies

DAPI was used to detect cell nuclei (blue). Both primary antibodies were omitted in the negative control (d, e, f). Bar, 10 μm; magnification 100x. Higher magnification of the boxed areas in (a′), (b′) and (c′); white arrowheads in (c′) indicate colocalization foci of ORF1 and ORF2 proteins.

Figure 3. Detection of ORF2 protein in human cancer cell lines

(A) Immunoblotting analysis of ORF2p in the indicated cell lines. WI-38, normal fibroblasts; A-375, melanoma; U-87, glioblastoma; HT-29, colon carcinoma; H69, small cell lung carcinoma; BxPC-3, pancreas carcinoma; LnCAP, PC-3 and DU145, prostate carcinoma cell lines (50 μg/lane). α-tubulin served as loading control. (B) Densitometric analysis of ORF2p signal intensity. The data (normalized to α-tubulin) are shown as mean ± S.D. of three independent experiments.

Figure 4. Immunohistochemical staining of ORF2p in human normal and cancer tissue sections

Representative tissue sections from: normal colonic mucosa (a), normal prostatic gland (c), normal lung epithelium (e), normal breast (g) and respective carcinomas (b), (d), (f), (h). (N), normal; (T), tumor tissue. Bar, 50 μm; magnification 20x. Boxed areas are shown at a higher magnification in (b′), (d′), (f′) and (h′). Arrow, nuclear localization; arrowhead, cytoplasmic localization.

Figure 5. Immunohistochemical staining of ORF2p in human colon tissue sections

(A) Representative tissue sections from: (a), (d) normal mucosa; (b) transition zone from normal (white arrowhead) to dysplastic mucosa (black arrowhead); (c) adenoma with medium grade dysplasia; (e) transitional mucosa; (f) adenocarcinoma; (T) indicates tumor cells expressing ORF2p, stroma adjacent to the tumor is indicated with (S). Bar, 50 μm; magnification 20x. (B) Scatter plot showing distributions and mean values ± SEM of signal scores of colon tissue specimens. Signal scores were assigned to every specimen as described in Materials and Methods; ***P < 0.001.

Figure 6. Immunohistochemical staining of ORF2p in human prostate tissue sections

(A) Representative tissue sections from: normal gland (a); prostatic intraepithelial neoplasia (PIN) (b); adenocarcinoma with Gleason pattern 3 (c), 4 (d) and 5 (e); intraluminal necrotic cells are indicated with (Ne). (N), normal tissue; bar, 50 μm; magnification 20x. (B) Scatter plot showing distributions and mean values ± SEM of signal scores of prostatic tissue specimens. *P < 0.05; **P < 0.01; ***P < 0.001.