The Murine Mammary Tumor Virus Revisited
Until the virological requirements to prove causation are fulfilled, the role of the MMTV-like retrovirus found in human cancers remains elusive.
Published April 1, 2003
By Beatriz G.-T. Pogo
Academy Contributor

Discovery that an agent in the milk of certain strains of mice was able to cause mammary tumor (Bittner, Science 84: 162 1936) led to the possibility that a similar agent may play a role in human breast cancer (BC).
Although numerous immunological and molecular findings have related the mouse mammary tumor virus (MMTV) to human breast carcinogenesis, the non-specificity of the immune reactions, the presence of retroviral sequences in the human genome with homology to MMTV, and lack of epidemiological results have weakened the evidences favoring such hypotheses. These controversial findings have been extensively discussed in a previous publication (Pogo and Holland 1997 Review in Biological Trace Element Research 56:131).
Following a new approach, we have re-investigated the participation of a retrovirus similar to MMTV. First, we looked for sequences in the MMTV genome that were not homologous to known human endogenous retrovirus (HERV) sequences. A 660-bp segment of the MMTV env gene with low homology to HERV- K10 (the HERV most similar to MMTV) was identified, and then primers were designed for use in amplification techniques such as polymerase chain reaction (PCR).
Characterizing the Phenotype of the Env-Positive Tumors
Secondly, the presence of the env sequence was investigated in a panel of human BC directly obtained from the operating room to avoid the necessity of using cell lines whose origins and maintenance might be questionable. The results indicated that the 660-bp sequence was present in 38 percent of the 500 tumors examined from the Mount Sinai Hospital in New York.
DNA from 100 normal breasts obtained from reduction mammoplasty and from other normal organs was negative. Cloning and sequencing of the human amplified sequences revealed that they were 90-98 percent homologous to MMTV, but not to HERV-K10 (Wang et al 1995 Cancer Research 55:5173). The 660-bp sequence was also expressed as RNA in 66 percent of the tumors that were env-sequence positive (Wang et al 1998 Cl Cancer Research 4:2565-2568).
To characterize the phenotype of the env-positive tumors, a study was conducted in collaboration with researchers from the Istituto Nazionale per lo studio e la cura dei Tumori in Milan, Italy. From studies of clinical, pathological and molecular parameters, the only association found with env positivity was the expression of laminin receptor, a marker for malignancy and bad prognosis (Pogo et al 1999 Cl Cancer Research 5: 2108). The laminin receptor has been recognized as a receptor for certain viruses. It is also significant that in a group of very aggressive breast tumors, like the gestational breast cancer, the viral sequences were detected at a higher (67.5) percentage (Wang et al 2003, unpublished results).
Endogenous or Exogenous in Origin?
The question of the endogenous or exogenous origin of the sequences was addressed by analyzing BC and normal breast tissues from the same patient, using samples obtained from the Breast Cancer Registry of the National Cancer Institute (NCI). The results demonstrated that of 106 BC and 106 normal breast samples studied, 30 percent of the BC – but just one normal sample – was positive for the env gene sequence. These findings suggest that the MMTV-like sequences are exogenous, since the possibility of polymorphism of the endogenous sequences was ruled out (Melana et al 2000 Cl Cancer Research 7:283-284).
Presence of other viral genes was investigated by successive PCR amplifications. A whole proviral structure, highly homologous to MMTV, was detected in two independent breast tumors. The 9.9-kb proviral structure displays all the features of a replicative, competent retrovirus, which is 95 percent homologous to MMTV, but only 57 percent to HERV K-10 over the gag and pol domains. By fluorescence in vitro (FISH) hybridization, a 2.7-kb env-LTR sequence was found integrated into several chromosomes of env-positive breast cancer cells. The 2.7-kb sequence was absent in two normal breast cell lines studied (Liu et al 2000 Cancer Research 61:1754-1759).
The findings described above lead us to investigate the presence of viral particles in primary cultures of human BC cells. Preliminary experiments used primary cancer cell cultures obtained directly from pleural or ascites effusions of patients whose BC contained env sequences. They revealed the presence of retroviral particles in the culture media, as detected by reverse transcriptase (RT) activity. These particles show morphological and sedimentation characteristics of a retrovirus. Amplification and sequencing of the c-DNA obtained from isolated particles indicated the presence of viral genes homologous to MMTV (Melana et al 2001 Proc AACR 42:115).
Experiments in Progress
Experiments in progress are aimed to explore if these particles can replicate, infect and transform cells in vivo and in vitro. Until these virological requirements to prove causation are fulfilled, the role of the MMTV-like retrovirus found in human cancers remains elusive.
Our findings have been reproduced independently by Etkind et al – who also have extended these studies to lymphomas (Etkind et al 2000 Cl Cancer Research 6:1273) – and by Ford et al in Australian and Vietnamese women (Ford et al Cl Cancer Research in press).
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About the Author
Beatriz G.-T. Pogo, professor of Medicine (Division of Hematology Oncology) and Microbiology, The Mount Sinai School of Medicine, New York University.