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.     

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.     

The mouse mammary tumor virus-like env gene sequence is not detectable in breast cancer tissue of Austrian patients

The mammary mouse tumor virus (MMTV) has been related to human breast cancer in previous studies, but these have yielded contradictory results. An MMTV env gene-like sequence was detectable in a relatively high proportion (38%) of human breast cancer tissues. The aim of this study was to determine the proportion of this 660 bp MMTV env gene-like sequence in a population of Austrian breast cancer patients. We performed PCR, repeat PCR, and nested PCR. We did not find any exogenous MMTV env gene sequences in the 50 DNA samples of human breast cancer tissue nor in 22 breast cancer cell lines including MCF-7, which has previously been described as a positive control.     

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.     

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.     

Clonal isolation of different strains of mouse mammary tumor virus-like DNA sequences from both the breast tumors and non-Hodgkin's lymphomas of individual patients diagnosed with both malignancies.

PURPOSE: In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin's lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin's lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail. EXPERIMENTAL DESIGN: DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin's lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced. RESULTS: MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin's lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree. CONCLUSIONS: The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin's lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.     

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.     

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.     

自发Basal-like乳腺癌小鼠模型研究

探讨TA2小鼠自发乳腺癌生物学行为、形态及免疫表型特征,比较其与人Basal-like乳腺癌的相似程度,研究TA2小鼠乳腺癌发生与妊娠及小鼠乳腺癌病毒（mouse mammary tumor virus,MMTV）的关系。方法:收集TA2自发乳腺癌小鼠84只,采用H&E染色和免疫组织化学染色检测TA2自发乳腺癌组织形态、免疫表型与转移情况,比较与人Basal-like乳腺癌的相似程度。电化学法检测TA2自发乳腺癌小鼠（n=10）、妊娠15天小鼠（n=6）与正常小鼠（n=6）血清雌二醇和孕酮水平。电镜观察肿瘤细胞内是否存在MMTV病毒颗粒,使用PCR和Real-time PCR分别检测MMTV LTR在TA2正常小鼠乳腺、妊娠小鼠乳腺和乳腺癌中基因组与mRNA表达变化。结果:TA2自发乳腺癌小鼠平均见瘤年龄（329.81±95.32）天,平均分娩次数（2.70±1.82）次,且肿瘤在妊娠期内迅速增长。肿瘤多发生内脏转移,淋巴结转移未见,80.0%（64/80）发生肺转移。TA2自发乳腺癌形态一致,癌巢由胞浆稀少的小圆细胞构成,周围被发育良好的基质包绕。实性癌巢中央为未分化或分化差的癌细胞,且核分裂像多见,中央可见坏死;癌巢边缘的癌细胞多发生分化,呈腺泡状,腺管状或乳头状;腺泡状癌巢周围有单层或多层肌上皮和致密基质包绕;腺管状分化癌巢中央可见粉色分泌物积聚;实性癌巢中央可见坏死。免疫组化染色显示TA2自发乳腺癌不表达ER、PR和HER2,高表达Cyclin D1、PCNA、p53和CK5/6。电镜观察显示肿瘤细胞内存在MMTV病毒颗粒。TA2自发乳腺癌小鼠血清雌二醇和孕酮浓度及瘤组织内MMTV LTR mRNA表达均升高,其改变与妊娠小鼠相似。结论:TA2小鼠自发乳腺癌形态、免疫表型和生物学特征与人Basal-like乳腺癌极为相似,能够反映Basal-like乳腺癌发生发展的全过程,是研究Basal-like乳腺癌的良好模型。妊娠激素水平升高导致的MMTV激活在Basal-like乳腺癌发生过程中起重要作用。      

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.     

Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

Background

The diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members.

Results

MMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own.

Conclusion

The presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice.     

Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.     

Mouse mammary tumor virus-like gene sequences in breast tumors of Australian and Vietnamese women.

PURPOSE: There is considerable evidence that the presence of mouse mammary tumor virus (MMTV)-like gene sequences in human breast cancer is highly associated with human breast carcinoma. Previous studies have found MMTV-like gene sequences in 38% of breast cancer tissue from United States women. The prevalence of these sequences in Australian and Vietnamese women has never been reported. EXPERIMENTAL DESIGN: Using PCR and primers that amplify MMTV-like gene sequences, we tested cancerous and benign breast tissue from Caucasian-Australian, Vietnamese-Australian, and Vietnamese women. RESULTS: MMTV-like gene sequences were amplified in 19 of 45 (42.2%) archival breast cancer biopsy tissues from Caucasian-Australian women, but only 1 of 120 (0.8%) and 0 of 41 breast cancer biopsy tissues from Vietnamese and Vietnamese-Australian women, respectively. The same sequences were found in only 2 of 111 (1.8%) and 0 of 60 normal (benign) breast tissue samples from Australian and Vietnamese women, respectively. CONCLUSIONS: MMTV-like gene sequences are found in only some human populations and are rarely found in normal human breast tissue from all populations, suggesting they are not present in the normal human genome and have been acquired.