Human saliva as route of inter-human infection for mouse mammary tumor virus

Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma

ISOLATION AND CHARACTERIZATION OF HUMAN MAMMARY TUMOR VIRUS (HMTV) IN HUMAN SPORADIC BREAST CANCER

Breast cancer is the most common cancer in women and in the 35-55 age range is the principal cause of death amongst women worldwide. It has become clear that the clinical onset of breast cancer is influenced by several factors. Among these, the potential involvement of a virus of the family Retroviridae, the Human Mammary Tumor Virus (HMTV), has represented a major focus of investigation whose existence has been proposed and disputed for many years after the identification of the MMTV (Murine Mammary Tumor Virus). Several scientists were able to demonstrate the presence of HMTVenv sequence (HMTVes) in 30-40% of invasive human breast carcinomas and very recently our laboratory was able to prove that its presence is strictly associated with sporadic breast cancer progression. In the present study we decide to add more evidence to the possible viral etiology of human breast cancer by approaching the problem from different angles: 1) the fact that in hereditary breast cancer (HBC) inherited gene mutations are recognized as initiating event led us to hypothesize that in this group an involvement of an oncogenic virus is not expected. The presence of MMTVes was investigated in a group of HBC from patients hosting a BRCA1 or BRCA2 mutations as well as in a group of SBC, with the aim to prove this hypothesis. 2) we decided to investigate the frequency of HMTVes in a set of sporadic breast cancer (SBC) samples collected in three different and geographically distant countries such as Sardinia, Jordan and Australia. In the last population when possible we collected also for each SBC case, a normal and pre-invasive lesion in order to assess the presence of the virus before cancer onset. 3) because the infiltrative carcinoma of the breast initially spreads via lymphatics giving metastasis firstly to the axillary and internal mammary lymph nodes, it was interesting to analyze lymph nodes of HMTVes positive and negative (HMTVes+ and -) tumors. From each metastatic lymph node was microdissected the tumoral epithelial cell population and the lymphocytes. 4) a gene expression study, using specific breast cancer arrays and a panel of selected genes, was performed to assess the genetic background in SBC HMTVes+ and – tumors and matched normal. 5) because of the strong similarities between the human and the murine disease, the presence of the MMTV virus in all murine exocrine glands led us to investigate if we could find HMTVes also in human saliva and salivary glands. 6) we have created HMTVes+ breast cancer cell primary cultures, from fresh human breast cancer tissues collected from the surgery-room in order to isolate the virus and FISH, Electron microscopy, Immunocytochemistry and Western experiments were performed to confirm the presence of the virus. The results obtained from all experiments support our recent findings and also our new hypotheses. We were able to obtain only one HMTVes+ primary cancer cell line which seemed to be HMTV positive also by electron microscopy, FISH, immunocytochemistry and Western Blot analysis