Transcription profile of a human breast cancer cell line expressing MMTV-like sequences

  • Mariana Fernandez-Cobo1,

    Affiliated with

    • Stella M Melana1,

      Affiliated with

      • James F Holland1 and

        Affiliated with

        • Beatriz GT Pogo1, 2Email author

          Affiliated with

          Infectious Agents and Cancer20061:7

          DOI: 10.1186/1750-9378-1-7

          Received: 26 October 2006

          Accepted: 15 December 2006

          Published: 15 December 2006

          Abstract

          Background

          It has been postulated that inflammation caused by certain viruses might result in cancer. Recently, it was shown that childhood lymphoblastic leukemia, breast and ovarian cancers express an interferon-related signature, providing support for this notion. We have previously shown that 38% of the sporadic breast cancers contain MMTV-like env gene sequences. To find out if the presence and expression of MMTV-like sequences correlated with an inflammatory phenotype, we have compared the expression profile of two sublines of MCF-7 cells, one containing the MMTV-like sequences (env+), the other one lacking them (env-).

          Results

          The results indicated that there were 47 differentially expressed genes between the two sublines. Among 27 upregulated genes in the env+ cells there were 7 interferon-related genes, 5 TNF-connected genes and 2 TGFβ-related genes.

          Conclusion

          These results suggest that the env+ cells were most likely responding to an infectious agent, and support the hypothesis that a viral infection may play a role in breast cancer pathogenesis.

          Background

          We and others [14] have shown that 37 to 41% of sporadic breast cancer samples contain MMTV-like env gene sequences. The sequences are expressed as RNA [5] and as protein in breast cancers (Melana et al., submitted). They are absent from the normal breasts of patients with env positive tumors [6] and are expressed as RNA exclusively in the cancer cells [7]. The whole proviral structure, designated human mammary tumor virus (HMTV), which has 95% homology to MMTV, can be detected in two tumors [8]. Although sequence variations are observed in the C-terminal of human sag sequences, the cloned human sag sequences expressed in human B lymphocytes can activate human T-cells, as can the mouse Sag, indicating that it can be functional [9]. Moreover, viral particles with the morphological characteristics of betaretroviruses are observed in primary cultures of human beast cancer [10]. Taken together, these results suggest that an infectious agent is present in some human breast cancers.

          Chronic inflammation has been implicated in tumor progression. New evidence suggests that the inflammation caused by certain viruses results in cancer [Reviewed in [11]]. Recently, it was reported that childhood lymphoblastic leukemia, as well as breast and ovarian cancers express an interferon-related signature, but not found in other human cancers studied [12]. This finding provides molecular support for the role of inflammation or viral infection in cancer pathogenesis [12].

          The established breast cancer cell line MCF-7 is widely used in research, and many subclones are available. Some of the original isolates produce retroviral-like particles [13]. Furthermore, May and Westerly [14] described the presence of an MMTV-like 6.6 Kb EcoR1 fragment in some of the MCF-7 cell lines, which was absent in other breast cancer lines and in normal tissue.

          Continuous passage with subsequent chromosomal change [15] may have eliminated viral sequences from some of them. It has been reported that some sublines of MCF-7 show biological differences [16] and significant genetic variation in RNA expression [17, 18].

          We have previously reported that a subline of MCF-7 containing env and LTR sequences [19, 20] and that it expressed the env gene as RNA [7], while other sublines were negative for env gene [[21] and our own results]. To find out whether the presence of viral sequences is related to an interferon-related signature, we have compared the expression profiles of two sublines of MCF-7 [22], one which contains the MMTV-like env gene sequence (env+) and one which lacks it (env-).

          Results

          The presence of the Env protein was investigated in both sublines. In Fig. 1 the result of the immunoblotting experiment is shown. The HMTV env+ cell line expressed a protein of a MW of approximately 50 kD which reacted with mAbP2, a monoclonal antibody against a synthetic peptide derived from human env sequences (Melana et al submitted). It was absent in the HMTV env- cells. Tubulin was equally present in both extracts.
          http://static-content.springer.com/image/art%3A10.1186%2F1750-9378-1-7/MediaObjects/13027_2006_Article_7_Fig1_HTML.jpg
          Figure 1

          Western blot of MCF-7 cells. Experimental conditions as described in Materials and Methods. A: MCF-7 (+) cells; B: MCF-7(-) cells.

          The results of the cDNA arrays are shown in Tables 1 and 2. Nineteen genes showed a > 2.5 fold difference in their adjusted intensity between HMTV env+ and env- cells, while another eight genes were only expressed in the HMTV env+ cells (Table 1). Twenty genes were downregulated (Table 2) in HMTV env + cells. Taken together, there were 47 differentially expressed genes. Among the 27 upregulated genes there were six interferon-inducible ones: IFI6, TRIM22, IFITM1, IFITM2+IFITM3, IFI27 and IP-30 and a receptor IFNGR2. In addition, there were five upregulated genes that have a connection with TNF or are involved in its signaling, like LTBR, TRAF3, MMP17, PKN1 and MAPK13. The cytokine TGFβ, and its downstream effector early growth response protein 1 (EGR1), were also upregulated in env+ cells. Twenty genes were down regulated in HMTV env+ cells.
          Table 1

          Up-regulated genes in HMTV env+ cells

          Acc #

          Gene symbol

          Protein/gene

          Ratio

          Diff

          X02492

          IFI6

          Interferon-inducible protein 6

          12.44

          4108

          J05633

          ITGB5

          Integrin beta 5

          9.46

          3401

          X82200

          TRIM22

          Tripartite motif-containing 22

          5.76

          1495

          L03840

          FGFR4

          Fibroblast growth factor receptor 4

          5.72

          1742

          J04164

          IFITM1

          Interferon induced transmembrane protein 1 (9–27)

          5.38

          6776

          M64595

           

          small G protein

          4.74

          1720

          X57351

          IFITM2 + IFITM3

          interferon induced transmembrane prot 2 (1–8D) + 3 (1–8U)

          4.25

          1082

          X89576

          MMP17

          matrix metalloproteinase 17

          4.20

          1647

          X52541

          EGR1

          early growth response protein 1

          3.39

          1327

          L29220

          CLK3

          CDC-like kinase 3

          3.38

          1521

          X66362

          PCTK3

          PCTAIRE protein kinase 3

          3.34

          1368

          U33053

          PKN1

          protein kinase N1

          2.85

          1075

          L04270

          LTBR

          lymphotoxin beta receptor (TNFR superfamily, member3)

          2.79

          1406

          U09579

          CDKN1A

          cyclin-dependent kinase inhibitor 1A (p21, Cip1)

          2.69

          6193

          U14966

          RPL5

          60S ribosomal protein L5

          2.68

          1641

          M29039

          JUNB

          jun-B

          2.65

          1313

          M29971

          MGMT

          6-O-methylguanine-DNA methyltransferase

          2.63

          1971

          M65199

          EDN2

          endothelin 2

          2.58

          1535

          U57342

          MLF2

          myeloid leukemia factor 2

          2.52

          2897

          X69398

          CD47

          CD47 glycoprotein; integrin-associated protein

          Up

          4152

          U12255

          FCGRT

          Fc fragment of IgG, receptor, transporter

          Up

          2960

          AF004709

          MAPK13

          mitogen-activated protein kinase 13

          Up

          2049

          X02812

          TGFB

          transforming growth factor, beta 1

          Up

          1535

          U21092

          TRAF3

          TNF receptor-associated factor 3

          Up

          1407

          U05875

          IFNGR2

          interferon gamma receptor 2

          Up

          1372

          X67325

          IFI27

          interferon, alpha-inducible protein 27

          Up

          1182

          J03909

           

          gamma-interferon-inducible protein; IP-30

          Up

          1076

          Table 2

          Down-regulated genes in HMTV env+ cells

          Acc #

          Gene symbol

          Protein/gene

          Ratio

          Diff

          U02687

          FLT3

          fms-related tyrosine kinase 3

          -6.20

          -2143

          X74295

          IGA7B

          integrin alpha 7B

          -5.13

          -2249

          X53587

          ITGB4

          integrin beta 4

          -4.80

          -2384

          M34671

          CD59

          CD59 molecule, complement regulatory protein

          -3.60

          -2551

          L25081

          RHOC

          ras homolog gene family, member C

          -3.59

          -2341

          U89278

          PHC2

          polyhomeotic-like 2

          -3.53

          -1250

          X16277

          ODC1

          ornithine decarboxylase 1

          -2.87

          -1295

          AF029670

          RAD51C

          RAD51 homolog C

          -2.61

          -1713

          M20430

          HLA-DRB1

          MHC class II HLA-DR-beta

          Down

          -4288

          J04111

          JUN

          c-jun proto-oncogene; transcription factor AP-1

          Down

          -1745

          U70310

          FANCG

          DNA repair protein XRCC9

          Down

          -1514

          M59911

          ITGA3

          integrin alpha 3

          Down

          -1393

          M97934

          STAT2

          signal transducer and activator of transcription 2

          Down

          -1248

          L38518

          SHH

          sonic hedgehog

          Down

          -1183

          X51521

          VIL2

          ezrin; villin 2

          Down

          -1177

          L07515

          CBX5

          chromobox homolog 5; heterochromatin protein homolog 1 (HP1)

          Down

          -1153

          M15400

          RB1

          retinoblastoma 1

          Down

          -1109

          M54995

          PPBP

          pro-platelet basic protein

          Down

          -1100

          X54199

          GART

          trifunctional purine biosynthetic protein adenosine 3

          Down

          -1092

          U47686

          STAT5 A +B

          signal transducer and activator of transcription 5 A+B

          Down

          -1072

          Discussion

          Comparison of the expression profiles of sublines derived from the same cell line provides an excellent model with minimal differences. Karyogenetic analysis revealed that the two sublines have similar complex chromosomal patterns (not shown). The comparison of expression profiles of MCF-7 env+ and env- cells indicated preferential expression of interferon-related genes: 26% (7/27) of the up-regulated genes. These differences may indicate a trend. Einav et al. [12] have reported that 40% of clinical breast cancer samples display an interferon-associated signature; 17 out of 36 (47%) of the upregulated genes. Our results are consistent with, but cannot be directly compared with those of Einav's for several reasons: we used only one cell line for analysis, the participation of stroma and surrounding tissues has been eliminated from our study, and finally, we used a different set of arrays. Nevertheless, our results strongly indicate that HMTV env+ MCF-7 cells express more interferon-related genes than the HMTV env- MCF-7 cells, suggesting that they may be responding to an infectious agent as proposed by Einav et al. [12]. The expression profile of HMTV env+ cells suggests an increased potential for cell growth, a fact that may be related to their more malignant phenotype as has been described in breast cancer cells associated with HMTV [23, 3, 24]. It is remarkable that the alpha 7 and beta 4 integrins were significantly down regulated in env+ cells, as has been reported in a set of finite life-span metastatic breast cancer cells which were also env+ [25].

          Whether the HMTV works as initiator and/or as promoter of malignant growth is uncertain. Molecular evidence that HMTV expression is responsible for the increase in interferon-related expression is being sought.

          Conclusion

          The results clearly indicate that the transcriptional profile of the cells expressing HMTV sequences is enriched in genes involved in inflammation process. This finding is significant because it was obtained comparing cells derived from the same cell line that have similar genetic background and minimal expressing differences. This supports the hypothesis that a viral infection may play a role in breast cancer pathogenesis.

          Methods

          MCF-7 cells were obtained from American Type Culture Collection (ATCC) and were propagated in vitro as recommended by the provider and as described in previous publications (1, 5). To determine whether the viral protein was expressed in our MCF-7 cells, western blotting was used. Protein lysates were prepared from approximately 1 × 107 cells. Equal amounts of protein from each sample were loaded onto an SDS-PAGE-10% polyacrylamide gel, followed by transfer to PVDF membranes. Western blot analysis was performed using mAbP2 (a monoclonal antibody against a peptide of the Env protein), and mAb-tubulin as primary antibodies (Sigma Aldrich). Proteins were visualized using horseradish peroxidase-labeled sheep anti-mouse IgG (GE Healthcare Bio-Sciences Corp.) as a secondary antibody followed by enhanced chemiluminescence (GE Healthcare Bio-Sciences Corp.).

          The expression profile was studied using the Atlas Human Cancer 1.2 cDNA expression array; a nylon membrane printed with 200–600 bp long fragments of 1176 characterized genes involved in cancer, 9 housekeeping genes and 6 negative controls (Clontech, CA). These conditions were described in detail in a previous publication [25]. Briefly, RNA was extracted and labeled with Atlas pure total RNA labeling system and hybridized to an Atlas Human Cancer 1.2 cDNA expression array (Clontech, CA) according to the manufacturer's instructions. Both cell sublines were probed twice in separate assays, and the accuracy of each duplicate was assessed by Pearson's correlation coefficient based on the adjusted intensity of all genes spotted on the membrane.

          Hybridizations with 30 μg of total RNA were performed according to the manufacturer instructions. The hybridized membranes were exposed onto a phosphorimager screen and were read using a phosphorimager reader (Molecular Dynamics). The scanned images were aligned and analyzed using AtlasImage 2.01 software (Clontech). When averaging or comparing samples, the adjusted intensity signal was normalized using the global normalization mode featured in the software. We reported only those genes whose ratios of differential expression were 2.5-fold or more, or genes that were undefined for one type of sample, but were detected on the other. (Undefined genes are those whose intensity were below the signal threshold) In the later event, when we lack a numerical value for the ratio, it was defined as being "up" or "down". Furthermore, for each gene we stated the difference (diff) in adjusted normalized intensity between the two cell lines.

          Accession number (Acc#), gene symbol and protein or gene name are according to GeneBank.

          Declarations

          Acknowledgements

          Grant support: The T.J. Martell Foundation for Leukemia, Cancer and AIDS Research, The Jane Grinberg Memorial Fund, the Kash Family Foundation, and the Ellen Block Memorial Fund.

          We thank Cindy Hernandez for skillful technical assistance and Jennifer Hasa and Julia Roboz for editorial work.

          Authors’ Affiliations

          (1)
          Department of Medicine, Mount Sinai School of Medicine
          (2)
          Department of Microbiology, Mount Sinai School of Medicine

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          Copyright

          © Fernandez-Cobo et al. 2006

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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