Variable transcriptional activity of endogenous retroviruses in human breast cancer - PubMed (original) (raw)

Variable transcriptional activity of endogenous retroviruses in human breast cancer

Oliver Frank et al. J Virol. 2008 Feb.

Abstract

Human endogenous retroviruses (HERVs) account for up to 9% of the human genome and include more than 800 elements related to betaretroviruses. While mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice, the role of retroviral elements in human breast cancer remains elusive. Here, we performed a comprehensive microarray-based analysis of overall retroviral transcriptional activities in 46 mammary gland tissue specimens representing pairs of nonmalignant and tumor samples from 23 patients. An analysis of nonmalignant tissue samples revealed a distinct, mammary gland-specific HERV expression profile that consists of 18 constitutively active HERV taxa. For corresponding tumor samples, a general trend toward lower levels of HERV transcription was observed, suggesting common regulatory mechanisms. In various subsets of patients, however, increased transcript levels of single class I HERV families (HERV-T, HERV-E, and HERV-F) and several class II families, including HML-6, were detected. An analysis of transcribed HML-6 sequences revealed either the activation of some or the increased activity of several proviral loci. No evidence for MMTV or human MMTV-like virus transcripts was found, indicating that transcriptionally active, MMTV analogous, exogenous viruses were not present in the breast cancer samples analyzed.

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Figures

FIG. 1.

FIG. 1.

Alignment of human MMTV-like (HML) DNA chip capture probes and demonstration of microarray discriminatory power. (A) Aligned nucleotide sequences of MMTV, HMLV, and endogenous HERV-K-related proviral elements (HML taxa) correspond to a 90-bp stretch of genomic DNA flanked by sequences encoding two highly conserved amino acid motifs (VLPQG and YV/MDDI/V/L, not included) commonly found in retroviral pol(RT) genes. See references and for the origins and classifications of HERV chip capture probes and amplification primers. Synthetic oligonucleotide probes, complementary to HERV-K(HML-1), HERV-K10 (HML-2 family), HERV-K(HML-5), MMTV, and HMLV capture probes used for optimization of the discriminatory power of the microarray assay, are underlined. (B) Specific hybridization signals were obtained employing Cy3-modified synthetic hybridization probes specific for MMTV (row 1), HMLV (row 2), and HML-1, HERV-K10 (HML-2 family), and HML-5 (row 3). Sensitive detection of MMTV was demonstrated by analyzing 100 ng of total RNA derived from murine GR cells in a standardized retrovirus DNA chip assay (row 4). Asterisks denote capture probe sequences aligned in panel A. Ubiqui, ubiquitin; GAPD, glyceraldehyde 3-phosphate dehydrogenase; α-Tub, alpha tubulin; β-Act, beta actin.

FIG. 2.

FIG. 2.

HERV transcriptional activity in human breast cancer. A total of 46 samples representing pairs of normal (N) and malignant (T) mammary gland tissue specimens from the same patient (n = 23) were compared by microarray hybridization. A housekeeping gene panel served as an internal control. Cy3-labeled DNA hybridization probes were generated by RT-PCR on mRNA according to the standardized protocol (41). HERV elements representing the mammary gland-specific HERV activity profile are indicated by red boxes (bottom line). QRT-PCR was performed for a subset of four differentially active HERV elements (HERV-FRD, RT244, HERV-K-T47D, and Seq56), shown in red letters. For detailed information about the identity of targets and capture probes see references and . Albeit false color mapping was used for image visualization, the printed image may not properly display signal gradation, especially for weak signals.

FIG. 3.

FIG. 3.

Qualitative analysis of HERV transcription in pairs of mammary gland tissue specimens corresponding to nonmalignant and tumor cells of 23 breast cancer patients. For the determination of HERV incidences, only those HERVs with signal intensities above a defined cutoff value (4,000 relative units) were evaluated. Constitutively active HERVs are marked by black boxes (incidence, 22 or 23 out of 23), and differentially active (2 to 21 out of 23) and inactive (0 or 1 out of 23) HERV elements are depicted by gray and white boxes, respectively. Tumor-related changes in HERV incidences were defined by normalizing particular HERV transcript levels with their corresponding HPRT transcript levels, whereby only alterations greater than 10% were judged as changes. Asterisks denote HERV elements that were subjected to QRT-PCR. −, the P value was not significant after Fisher's exact test.

FIG. 4.

FIG. 4.

Quantitative analysis of HERV transcript levels based on microarray densitometry was performed for nine HERV elements, representing core components of the human mammary gland HERV transcriptome, and is illustrated by group (A) and pairwise comparisons (B). Based on mean values of triplicate DNA chip hybridizations, representative relative abundancies of HERV transcripts in each sample pair (N indicates normal tissue, and T indicates tumor tissue) were normalized by respective HPRT transcript levels. P values are given for median value comparisons of N and T groups using the Wilcoxon signed-rank test. N.s., not significant.

FIG. 5.

FIG. 5.

(A) Tissue type-specific (malignant versus nonmalignant) transcript level alterations of RT244(HML-3), HERV-K-T47D (HML-4 family), and HERV-FRD derived from DNA chip densitometry (upper panels) were compared with QRT-PCR data using subgroup-specific amplification primers (lower panels). (B) Analogous analysis of three HML-6 members [HERV-K(HML-6), Seq38, and Seq56]. Microarray analysis (upper panel) and QRT-PCR, using primers specific for the HML-6 family, according to the method of Muradrasoli and coworkers (33) (lower panel) was performed. Numbers above and below the bars correspond to identification numbers of analyzed patient samples. All ratio calculations (tumor [T] versus normal [N]) are based on values normalized to their respective housekeeping gene (HK6) levels [(HERV/HK6)T/(HERV/HK6)N] that were subsequently log2 transformed. Microarray and QRT-PCR data are based on triplicate experiments as outlined in Materials and Methods.

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