A mouse mammary tumor virus-like long terminal repeat superantigen in human breast cancer

We previously reported a 660-bp mouse mammary tumor virus (MMTV)-like env gene sequence in approximately 38% of human breast cancer DNA, but not in normal breasts or other tumors. This MMTV-like env gene sequence was expressed in 66% of the env gene-positive human breast cancers. An entire proviral structure was identified in human breast cancer DNA with high homology to MMTV and low homology to known human endogenous retrovirus. MMTV-like long terminal repeat (LTR) sequences were also detected in 41.5% of human breast cancers. They contain hormone-responsive elements, TEF-1 family elements, and the open reading frame for the superantigen (SAg). We have now amplified and sequenced MMTV-like sag sequences from 10 human breast cancers, and we found that they are highly homologous to those of MMTV. However, deletions and insertions at the COOH-terminal of sag were observed. The immune function of the human MMTV-like LTR SAg was also investigated. The sag gene was cloned and expressed in a human B-cell line (Ramos). T-cell proliferation and cytokine releasing assays were performed after cocultivation of T cells with irradiated Ramos SAg-expressing cells. The results indicate that expression of the human SAg stimulates T-cell activation in vitro, as the mouse SAg does. Because the T-cell responses in vitro are considered similar to those in vivo, these results suggest that the human LTR SAg might also play a role in human breast carcinogenesis.     

Prevalence and characteristics of the MMTV-like associated breast carcinomas in Tunisia

The involvement of a retrovirus homologous to the mouse mammary tumor virus (MMTV) in the pathogenesis of human breast cancer (BC) has long been assumed, but has never been proven. Previous studies have reported the detection of MMTV-like env sequences in variable proportions that did not exceed 40% of BC cases in several countries. However, these viral sequences have been found in higher proportion (74%) in Tunisian diagnosed with BC during the seventies. This study is an attempt to evaluate the current prevalence of MMTV-like env gene in BC in Tunisian women. We used semi-nested PCR that amplify a 190-bp MMTV-like env sequence, followed by direct sequencing to screen a series of 122 cases of BC randomly selected. The findings were correlated to clinicopathological data and immunohistochemical expression status of progesterone and oestrogen receptors, HER2, and P53. Specific MMTV-like env sequences were found in 17 (13.9%) cases of breast carcinomas, whereas the same sequences were not detected in matched normal breast tissues. The presence of the viral sequences correlates inversely with progesterone receptor expression (6.8% versus 20.3%; P=0.03) and HER2 overexpression (3.1% versus 17.7%; P=0.04). This present study confirms the presence of MMTV-like env sequences in BC in Tunisian women but describes an important decrease in the prevalence of the viral sequences compared with previous studies. This reduction may be due to some changes in the virological characteristics or exposure to the virus.     

No evidence of MMTV-like env sequences in specimens from the Australian Breast Cancer Family Study

Numerous independent groups from a range of countries have reported a high prevalence of Mouse Mammary Tumour Virus (MMTV)-like env sequences in human breast cancer specimens, including a prevalence of almost 40% in Australia. MMTV-like sag sequences and a completely integrated provirus have also been described. Recently, it was reported that MMTV is capable of productive infection of human breast cells in vitro. Conclusive demonstration of an association between MMTV and human breast cancer has remained elusive, and negative findings from a number of independent studies have questioned the role of MMTV as an aetiological agent. We used breast cancer specimens from women in the Australian Breast Cancer Family Study (ABCFS) who were diagnosed with first primary invasive breast cancer before the age of 40 years. Specimens were selected for higher grade cancers and for diagnosis relatively soon after childbirth. We searched for MMTV-like env sequences in tumour-enriched DNA using a nested PCR designed to detect all MMTV variants represented in GenBank, including those reportedly detected in human breast cancers. Forty-two specimens were deemed adequate for testing based on strong β-globin PCR. Despite the MMTV nested PCR regimen consistently detecting five copies of control plasmid against a background of MMTV-negative human genomic DNA, no MMTV env sequence was detected in any of the breast cancer specimens. Our findings appear inconsistent with previous reports on Australian breast cancer specimens but consistent with a growing number of independent negative reports internationally. We recommend caution in inferring a role for MMTV or a closely related virus in human breast cancer and suggest that universally regarded alternative lines of evidence such as highly specific serology data will be required to support such an association.     

Detection and identification of mouse mammary tumor virus-like DNA sequences in blood and breast tissues of breast cancer patients

Mouse mammary tumor virus (MMTV) is a well-known cause of mammary tumors in mice transmitted as endogenous proviruses or exogenously as infectious virions. The hypothesis that a retrovirus homologous to MMTV is involved in human breast cancers has resulted in renewed interest in the etiology of human breast cancer. Therefore, the detection of MMTV-like exogenous sequences in 30–40 % of invasive breast cancer has increased attention towards this hypothesis. To detect the prevalence of MMTV in Pakistani population, 666-bp-long MMTV envelop and 630-bp LTR sequences were amplified from breast cancer patient samples (tissue biopsies and peripheral blood) using mouse with mammary tumor as control. MMTV-like virus env and LTR DNA sequences were detected in 20 and 26 % of breast tumor samples, respectively, from the total of 80 breast cancer patients’ blood and tissue samples. No significant association was observed between age, grade of disease, and lymph node involvement with the prevalence of MMTV-like sequences. Our data add to the growing number of studies implicating MMTV-like virus in human breast cancer, but still clear causal association of MMTV to breast cancer remains to be reputable.     

Lack of Detection of the Mouse Mammary Tumor-like Virus (MMTV) Env Gene in Iranian Women Breast Cancer using Real Time PCR

Background: Mouse mammary tumor virus (MMTV) is the major cause of mammary tumors in mice.There is limited controversial evidence about the probable etiologic role of MMTV- like virus in human breastcancer. Materials and Methods: A total of 40 Formalin fixed paraffin embedded samples with diagnosis of breastcancer were collected in a period of 3 years from cancer institute of Iran. We selected both pre-menopausal andpost-menopausal patients with different histologic grades and different ethnic groups. We evaluated presenceof MMTV-like virus env gene through real time PCR method. Results: Forty patients (20 pre and 20 postmenopausalwomen) were evaluated with the mean age of 49.67. The average tumor size was 39 mm. None ofthe studied samples were positive for MMTV-like virus env gene target sequences. Conclusions: We found noevidence on the potential role of MMTV-like virus in the carcinogenicity of breast cancer among Iranian women.     

Mouse mammary tumor virus-like ENV gene sequences in human breast tumors and in a lymphoma of a breast cancer patient.

DNA sequences with very high similarity (95-98%) to the mouse mammary tumor virus (MMTV) ENV gene have been amplified by PCR in 38.5% of human breast tumors and in <2% of normal breast tissue (Wang et al., Cancer Res., 55: 5173-5179, 1995). Intrigued by these findings, which suggested an exogenous viral etiology for a certain percentage of human breast tumors, we have screened a panel of human breast tumors and normal breast tissue for the presence of MMTV-like DNA sequences. Using similar PCR procedures and stringent hybridization techniques, we have detected the presence of MMTV-like ENV gene sequences in 37% of the human breast tumors that we have analyzed. DNA sequencing has shown these sequences to be 99% homologous to the BR6 strain of MMTV and 100% homologous to the GR and C3H strains of MMTV. We have not detected these MMTV-like sequences in normal breast tissue. However, we have detected these sequences by PCR and stringent hybridization in a T-cell lymphoma of a breast cancer patient who was simultaneously diagnosed with both diseases. Our results support the possibility of an exogenous retroviral etiology for a certain percentage of human breast tumors. Our results also suggest that a similar exogenous retroviral etiology may exist for certain human T-cell lymphomas. In many inbred strains of mice, both breast cancer and T-cell lymphoma are caused by MMTV, hence, in a certain percentage of humans, one or both of these diseases may be caused by an MMTV-like retroviral entity.     

Stromal immunoglobulin κC expression is associated with initiation of breast cancer in TA2 mice and human breast cancer

The initiation of spontaneous breast cancer (SBC) in Tientsin Albino 2 (TA2) mice is related to mouse mammary tumor virus (MMTV) infection, and MMTV amplification is hormonally regulated. To explore the insertion site of MMTVLTR in TA2 mouse genome, reverse PCR and nested PCR were used to amplify the unknown sequence on both sides of the MMTV‐LTRSAG gene in SBC and normal breast tissue of TA2 mice. Furthermore, the clinicopathological significance of the insertion site was evaluated in 43 samples of normal breast tissue, 46 samples of breast cystic hyperplasia, 54 samples of ductal carcinoma in situ, 142 samples of primary breast cancer and 47 samples of lymph node metastatic breast cancer by RNA in situ hybridization. We confirmed that the insertion site of the MMTV‐LTRSAG gene was located between Ig κ v2‐112 and Ig κ v14‐111 in chromosome 6 of TA2 mouse. IG κ C was localized in the stromal cells of TA2 mouse with SBC and in human breast cancer tissues. Tumor cells were negative for IG κ C in RNA in situ hybridization. The positive staining index of IG κ C in stromal cells was the highest in lymph node metastatic breast cancer, followed by primary breast cancer, ductal carcinoma in situ, and breast cystic hyperplasia. Furthermore, the positive staining index of IG κ C was related to the expression of ER, PR, HER2 and Ki‐67. Our findings showed that stromal IG κ C expression was associated with the initiation of SBC in TA2 mice. IG κ C may be a high‐risk factor for the initiation and progression of human breast cancer.     

小鼠乳腺肿瘤病毒及其与乳腺癌的关系

小鼠乳腺肿瘤病毒(MMTV)分布较为广泛,可在许多近交系小鼠品系中诱发乳腺癌.近年来研究发现,在人乳腺癌组织中存在MMTV相似基因序列,并提出MMTV可以诱发人乳腺癌的假说.如果MMTV可诱发人乳腺癌,就可利用MMTV抗体治疗某些乳腺癌.MMTV诱发小鼠乳腺癌的发生相关机制以及该病毒能否引发人乳腺癌、MMTV通过何种途径进入人体,都是目前研究的热点.     

High prevalence of MMTV-like env gene sequences in gestational breast cancer

Gestational breast cancer (BC) is generally associated with rapid growth and increased mortality. Because the presence of MMTV-like sequences in BC has been associated with laminin receptor expression, a marker of poor prognosis, gestational BCs were analyzed for MMTV env gene-like sequences to explore whether MMTV-like sequences were also associated with its adverse outcome. Whereas 30–38% of sporadic BC have the sequences, in gestational BC the prevalence is 62%. We suggest that hormonal response elements present in the MMTV-like LTR may play a role in promoting cell growth, as they do in the mouse system.     

Search for mouse mammary tumor virus-like env sequences in cancer and normal breast from the same individuals.

We have reported previously that a 660-bp sequence homologous to the env gene of the mouse mammary tumor virus, but not to the known endogenous retroviruses, was present in 38% of human breast cancers (Y. Wang et al., Cancer Res., 55: 5173-5179, 1995). A unique 250-bp internal sequence was equally present in formalin-fixed breast carcinoma. It was not detected in normal human breasts or in other tumors. In this study, we have investigated whether this 250-bp env sequence was also present in the formalin-fixed normal tissues of individuals with env sequence-positive breast cancer. Separate paraffin-embedded sections from breast carcinoma and normal breast tissues from the same individual were obtained from the Cooperative Breast Cancer Tissue Registry of the National Cancer Institute. The 250-bp env sequence was detected in 30.1% of the 106 tumors but in only 1 of the 106 normal breast tissues. These results indicate that the sequence is absent in normal tissues and thus is not genetically transmitted. This strongly implies that it is of exogenous origin.     

Increasing evidence for a human breast carcinoma virus with geographic differences

BACKGROUND: An early immunologic study suggesting that a virus similar to the mouse mammary tumor virus (MMTV) was associated highly with breast carcinoma in Tunisian patients, compared with patients in the United States, led the authors to examine different breast carcinoma populations by using more current molecular techniques. METHODS: Thirty-nine paraffin blocks were selected for sequencing of the 250-base pair segment of the MMTV from patients with breast carcinoma who were seen and treated at the Institut Salah Azaiz in Tunisia. Fifteen of those blocks were examined under code by a second laboratory, which used a different methodology and was blinded to the results of the first laboratory, and 14 blocks were analyzed successfully. RESULTS: The comparison of Tunisian patients and patients from other countries clearly showed a significantly higher proportion of tumors with MMTV-like sequences in the Tunisian series of patients. There was complete reproducibility of data between the two laboratories. Using the results from the first laboratory and similar studies from the literature, detection of the MMTV-like env gene sequence showed an important geographic pattern with a significantly higher percentage of positive patients with breast carcinoma in Tunisia (74%) compared with patients with breast carcinoma in the United States (36%), Italy (38%), Australia (42%), Argentina (31%), and Vietnam (0.8%) CONCLUSIONS: The findings provided increased evidence for a human breast carcinoma virus with geographic differences in prevalence. The geographic differences were compatible with studies of MMTV in wild mice; thus, the data were plausible biologically.     

Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women

Mouse mammary tumor virus (MMTV)-like sequences have been found in up to 40% of breast cancer samples but in <2% of normal breast tissue samples from Australian women studied by our group. Screening of a larger and more diverse cohort of female breast cancer samples has now shown a correlation of MMTV-like sequences with the severity (grade) of breast cancer. Thirty-two percent (43 of 136) of female breast cancer samples were positive for MMTV-like sequences when screened using PCR. A significant gradient of MMTV positivity was observed with increasing severity of cancer from 23% of infiltrating ductal carcinoma (IDC) grade I tumors to 34% of IDC grade II tumors (P = 0.00034) and 38% of IDC grade III tumors (P = 0.00002). We also report for the first time the detection of MMTV-like sequences in 62% (8 of 13) of male breast cancer samples and 19% (10 of 52) of male gynecomastia samples screened. MMTV-like sequences were demonstrated in various premalignant breast lesions of females, including fibroadenoma (20%) and fibrocystic disease (28%) samples, at a significantly higher prevalence than that seen in normal breast tissue (1.8%; P = 0.00001). Study of a longitudinal cohort of female breast cancer patients indicated that MMTV was co-incident with tumor but was not present when tumor was absent on histology. These results support the association of MMTV-like sequences with development of breast tumors in men and women and suggest association of MMTV with increasing severity of cancer.     

Mouse mammary tumor-like virus is associated with p53 nuclear accumulation and progesterone receptor positivity but not estrogen positivity in human female breast cancer.

PURPOSE: The purpose is to compare the presence of proteins with known associations with breast cancer-progesterone receptor (PgR), estrogen receptor, and p53, with the prevalence of mouse mammary tumor virus (MMTV)-like DNA sequences in human female breast cancers. EXPERIMENTAL DESIGN: A cohort of 128 Australian female breast cancers were screened for MMTV-like DNA sequences using PCR. The presence of PgR, estrogen receptor, and nuclear accumulation of p53 protein was assessed in the same samples using immunohistochemical staining. RESULTS: Nuclear accumulation of p53 was significantly more prevalent (P = 0.05) in archival human breast cancers containing MMTV-like DNA sequences. The presence of progesterone receptor was significantly higher in MMTV-positive than MMTV-negative breast cancers (P = 0.01). No correlation between estrogen receptor and MMTV-like DNA sequences was found. CONCLUSIONS: MMTV causes breast cancer in mice, and hormones up-regulate expression of virus in mice mammary tissue. It is unknown if this is the case in human breast cancers shown to contain DNA of MMTV-like viruses. The positive association between MMTV-like DNA sequences and PgR indicates hormones and MMTV may play a role in human breast cancer. Mutations of the tumor suppressor gene p53 are common in human breast cancer and are associated with higher grades of cancer. The association of MMTV-like DNA sequences with higher grades of cancer, and the positive association between p53 and MMTV-like DNA sequences clearly warrant additional investigation.