Mouse Mammary Tumor Virus Research Articles

CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

BackgroundIn earlier studies we have shown that CCL5 activation of CCR5 induces the proliferation and survival of breast cancer cells in a mechanistic target of rapamycin (mTOR)-dependent manner and that this is in part due to CCR5-mediated increases in glycolytic metabolism.MethodsUsing the MDA-MB-231 triple negative human breast cancer cell line and mouse mammary tumor virus – polyomavirus middle T-antigen (MMTV-PyMT) mouse primary breast cancer cells, we conducted in vivo tumor transplant experiments to examine the effects of CCL5-CCR5 interactions in the context of regulating tumor metabolism. Additionally, we employed Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry imaging (MALDI-FTICR-MSI) to evaluate tumor utilization of cellular metabolites.ResultsWe provide evidence that, in the absence of CCR5, the early events associated with rapid tumor growth in the MMTV-PyMT mouse model of spontaneous breast cancer development, are diminished, as demonstrated by a delay in tumor onset. In tumor transplant studies into immunocompromised mice we identify a direct correlation between reduced tumor proliferation and decreased metabolic activity, specifically associated with tumor expression of CCR5. The reduction in tumorigenesis is accompanied by decreases in glucose uptake, glucose transporter-1 (GLUT-1) cell surface expression, intracellular ATP and lactate levels, as well as reduced CCL5 production. Using MALDI-FTICR-MS, we show that the rapid early tumor growth of CCR5+/+ triple negative breast cancer cells in vivo is attributable to increased levels of glycolytic intermediates required for anabolic processes, in contrast to the slower growth rate of their corresponding CCR5−/− cells, that exhibit reduced glycolytic metabolism.ConclusionsThese findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis.

MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs

Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17–92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology.

Clonal deletion of T cell repertoires with specific T cell receptor Vβ chains by two endogenous superantigens in NC/Nga mice.

Superantigens (SAgs) are powerful T-cell stimulatory proteins. Because an atopic dermatitis (AD) model NC/Nga mice had two endogenous SAgs, namely minor lymphocyte-stimulating locus-1a (Mls-1a) and mouse mammary tumor virus (MMTV)(SHN), SAg-responsive T-cells bearing Vβ5.1, Vβ6, Vβ8.1, Vβ8.2, Vβ8.3, Vβ9, and Vβ11 should be endogenously deleted. Here, we discuss that the endogenous SAgs-expression may be involved in AD-sensitivity in NC/Nga mice.

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation.

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus – Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT;ApcMin/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT;ApcMin/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;ApcMin/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

Multiple oncogenic viruses are present in human breast tissues before development of virus associated breast cancer

BackgroundMultiple oncogenic viruses including, mouse mammary tumor virus, bovine leukemia virus, human papilloma virus, and Epstein Barr virus, have been identified as separate infectious pathogens in human breast cancer. Here we demonstrate that these four viruses may be present in normal and benign breast tissues 1 to 11 years before the development of same virus breast cancer in the same patients.MethodsWe combined the data we developed during investigations of the individual four oncogenic viruses and breast cancer. Patients who had benign breast biopsies 1–11 years prior to developing breast cancer were identified by pathology reports from a large Australian pathology service (Douglas Hanly Moir Pathology). Archival formalin fixed specimens from these patients were collected. The same archival specimens were used for (i) investigations of mouse mammary tumour virus (also known as human mammary tumour virus) conducted at the Icahn School of Medicine at Mount Sinai, New York and at the University of Pisa, Italy, (ii) bovine leukemia virus conducted at the University of California at Berkeley,(iii) human papilloma virus and Epstein Barr virus conducted at the University of New South Wales, Sydney, Australia.Seventeen normal breast tissues from cosmetic breast surgery conducted on Australian patients were used as controls. These patients were younger than those with benign and later breast cancer.ResultsStandard and in situ polymerase chain reaction (PCR) methods were used to identify the four viruses. The detailed methods are outlined in the separate publications.: mouse mammary tumor virus, human papilloma virus and Epstein Barr virus (Infect Agent Cancer 12:1, 2017, PLoS One 12:e0179367, 2017, Front Oncol 5:277, 2015, PLoS One 7:e48788, 2012).Epstein Barr virus and human papilloma virus were identified in the same breast cancer cells by in situ PCR.Mouse mammary tumour virus was identified in 6 (24%) of 25 benign breast specimens and in 9 (36%) of 25 breast cancer specimens which subsequently developed in the same patients.Bovine leukemia virus was identified in 18 (78%) of 23 benign breast specimens and in 20 (91%) of 22 subsequent breast cancers in the same patients.High risk human papilloma viruses were identified in 13 (72%) of 17 benign breast specimens and in 13 (76%) of 17 subsequent breast cancers in the same patients.Epstein Barr virus was not identified in any benign breast specimens but was identified in 3 (25%) of 12 subsequent breast cancers in the same patients.Mouse mammary tumour virus 3 (18%), bovine leukemia virus 6 (35%), high risk human papilloma virus 3 (18%) and Epstein Barr virus 5 (29%) were identified in 17 normal control breast specimens.ConclusionsThese findings add to the evidence that multiple oncogenic viruses have potential roles in human breast cancer. This is an important observation because evidence of prior infection before the development of disease is a key criterion when assessing causation.

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

ABSTRACTMouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.

Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis

Heparanase is an endoglucuronidase that uniquely cleaves the heparan sulfate side chains of heparan sulfate proteoglycans. This activity ultimately alters the structural integrity of the ECM and basement membrane that becomes more prone to cellular invasion by metastatic cancer cells and cells of the immune system. In addition, enzymatically inactive heparanase was found to facilitate the proliferation and survival of cancer cells by activation of signaling molecules such as Akt, Src, signal transducer and activation of transcription (Stat), and epidermal growth factor receptor. This function is thought to be executed by the C-terminal domain of heparanase (8c), because over expression of this domain in cancer cells accelerated signaling cascades and tumor growth. We have used the regulatory elements of the mouse mammary tumor virus (MMTV) to direct the expression heparanase and the C-domain (8c) to the mammary gland epithelium of transgenic mice. Here, we report that mammary gland branching morphogenesis is increased in MMTV-heparanase and MMTV-8c mice, associating with increased Akt, Stat5 and Src phosphorylation. Furthermore, we found that the growth of tumors generated by mouse breast cancer cells and the resulting lung metastases are enhanced in MMTV-heparanase mice, thus supporting the notion that heparanase contributed by the tumor microenvironment (i.e., normal mammary epithelium) plays a decisive role in tumorigenesis. Remarkably, MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands. Although this occurs at low rates and requires long latency, it demonstrates decisively the pro-tumorigenic capacity of heparanase signaling.

The Wnt/Β-catenin Signalling Pathway Inhibitor Sclerostin is a Biomarker for Early Atherosclerosis in Obesity

Sclerostin is an inhibitor of the wingless-type mouse mammary tumor virus integration site family/β-catenin signalling pathway (WβcSP), which plays an important role in bone metabolism and in vascular biology. It could act protective regarding atherosclerosis development through its effect on WβcSP in vascular cells. Nevertheless, results of studies analyzing association between circulating sclerostin level (CSL) and atherosclerotic diseases (AD) are showing conflicting results. The aim of this study is to test the value of CSL as a biomarker of subclinical carotid atherosclerosis (SCA) in obese persons. The cross-sectional study included 50 obese persons without previous history of diabetes and AD. Participants underwent adequate anthropometrical, ultrasound and laboratory examinations, including 2h 75 g oral glucose tolerance test (OGTT). Only the presence of SCA significantly indirectly correlated with CSL (p&lt;0.05). Based on the median value of CSL, we formed two groups: low CSL (CSL&lt;7.9 pmol/l) and high CSL (CSL&gt;7.9 pmol/l). There were no statistically significant differences in general (gender, age and current smoking) and anthropometrical characteristics (body mass index, waist circumference, systolic and diastolic blood pressure), inflammatory (total white blood cell count, erythrocyte sedimentation rate, fibrinogen, C-reactive protein and uric acid), glucose metabolism (fasting and 2h OGTT blood glucose, glycated hemoglobin and presence of dysglycemia) and lipid metabolism (low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B and lipoprotein (a)) parameters between low and high CSL groups. Low CSL group had significantly higher incidence of SCA (p&lt;0.05). CSL could serve as a useful biomarker of early atherosclerosis in obese persons without previous history of cardiometabolic disorders but the final conclusion requires further testing.

Moderate sensitivity of mouse mammary tumour virus to inhibition by human APOBEC3G.

Infectivity of the mouse mammary tumour virus (MMTV) is inhibited by mouse APOBEC3 (mA3) which is efficiently packaged into virions. As the inhibition is only partial, the virus can replicate in tissues expressing mA3 and complete its replication cycle. Here, we have examined the sensitivity of MMTV to inhibition by a human orthologue of mA3, A3G. We report that the virus containing A3G is only moderately susceptible to inhibition by the human factor. Whereas the vif-deficient HIV-1 vector produced in human epithelial cells expressing endogenous levels of A3G was efficiently inhibited, an MMTV vector remained fully infectious. Greater A3G expression levels were necessary to restrict infectivity of MMTV, but only when the factor retained its deaminase activity. Furthermore, the spreading kinetic of a replication competent MMTV was only moderately accelerated in cells with downmodulated A3G expression. These data suggest that MMTV has evolved a mechanism to neutralize antiviral activity of APOBEC3 proteins.

Exome sequencing reveals a novel homozygous splice site variant in the WNT1 gene underlying osteogenesis imperfecta type 3

BackgroundOsteogenesis imperfecta (OI) is a heritable bone fragility disorder usually caused by dominant variants in COL1A1 or COL1A2 genes. Over the last few years, 17 genes including 12 autosomal recessive and five autosomal dominant forms of OI, involved in various aspects of bone formation, have been identified.MethodsWhole-exome sequencing followed by conventional Sanger sequencing was performed in a single affected individual (IV-3) in a family.ResultsHere, we report the clinical and genetic characterization of OI type 3 in a consanguineous family with four affected members. Clinical examinations revealed low bone density, short stature, severe vertebral compression fractures, and multiple long bone fractures in the affected members. Exome sequencing revealed a biallelic pathogenic splice acceptor site variant (c.359-3C>G) in a wingless-type mouse mammary tumor virus integration site family 1 (WNT1) gene located on chromosome 12q13.12.ConclusionWe report a biallelic splice site variant underlying OI type 3 and the first case from the Pakistani population.

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy.

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

Prevalence of EBV, HPV and MMTV in Pakistani breast cancer patients: A possible etiological role of viruses in breast cancer

BackgroundBreast cancer being a multifactorial disease, the role of infectious agent in development of disease is of great interest. The high incidence of breast cancer around the world has woken the interest in a viral etiology of breast cancer. Despite decades of research, no etiologic factor(s) for human breast cancer has been known and the quest for a contributing cause has all but been abandoned during the past years. Recent investigations have linked breast cancer to viral infections, such as Epstein–Barr virus (EBV), Human papillomavirus (HPV) and mouse mammary tumor virus. AimTo investigate the possible association of EBV, HPV and MMTV infection with breast cancer development and progression. MethodsScreening of isolated genomic DNA from FFPE breast cancer tissue biopsies (n=250) using standard polymerase chain reaction and correlation of virus prevalence with BC disease outcomes using statistical analysis software (SPSS 16.0). ResultsOur findings suggest the prevalence of EBV (24.4%), HPV (18.1%) and MMTV (29.3%), while coinfection of HPV and EBV was detected in 9.2% (23/250), co infection of HPV and MMTV in 3.2% (8/250) and coinfection of EBV and MMTV in 6% (15/250) of breast cancer samples. No virus was detected in 59.5% of the breast cancer samples. Mono infection of EBV and HPV do not statistically co-relate with the clinico-pathological outcomes of breast cancer disease, though MMTV infection does co-relate with age and grade of breast cancer disease. In our study, the prevalence of coinfection of HPV, EBV and MMTV in Pakistani breast cancer patients is rare, still there is a possibility of synergistic carcinogenic effect of different viruses in the development of breast cancer disease. ConclusionThe significant percentage of virus prevalence shows potential role in breast cancer development. However, this study provides substantial but not conclusive evidence for the involvement of viruses in BC disease development and progressiveness.

Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents

Recent studies documented the detection of viruses strongly associated with human cancer in urban sewages and other water environments worldwide. The aim of this study was to estimate the occurrence of human oncogenic viruses in environmental samples (sewage, river, marine, and pool/spa water) using highly sensitive and specific multiplex bead-based assays (Luminex technology). A total of 33 samples were analysed for 140 oncogenic viral agents, including mucosal and cutaneous human papillomaviruses (HPVs), human polyomaviruses (HPyV), human herpesviruses (HHV) and mouse mammary tumour virus (MMTV).Eighty-eight percent of the samples tested positive for at least one viral pathogen and the simultaneous presence of more than one virus was frequent (mean number of positivities/sample = 3.03). A total of 30 different Alpha, Beta and Gamma HPVs were detected, including mucosal and cutaneous types. The high-risk type HPV16 was the most frequently detected virus, identified in 73% of the samples. Of the 12 HPyVs tested, only two (BKPyV and MCPyV) were detected. At least one of these two was present in 48% of the samples. MMTV was detected in 21% of the samples, while herpesviruses - HHV-6 and HHV-1 - were detected in two samples (6%).The present study is the first to provide a comprehensive picture of the occurrence of oncogenic viruses belonging to different families and species in diverse water environments, and the first to successfully use, in environmental samples, a Luminex-based multiplex platform for high throughput screening of infectious agents. Our findings, showing that oncogenic viruses are ubiquitous in water environments, pave the way for future studies on the fate of these pathogens in water environments as well as on their potential for transmission via the waterborne route.

Abstract 1844: Investigating the role of amphiregulin in breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG) is a ligand for EGFR that mediates estrogen-induced mammary ductal morphogenesis during development. Expression of AREG is enriched in invasive breast carcinomas, particularly ERα-positive breast tumors. Studies have shown that AREG increases the in vitro invasiveness of breast cancer cells. Furthermore, suppression of AREG expression in transformed human breast epithelial cells reduces tumor formation when injected in nude mice. These observations suggest that AREG plays an important role in breast cancer development and progression. To investigate the role of AREG in breast cancer development and progression, we utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors. Tumors in AREG KO PyMT mice initially grow more quickly but eventually reach 2 cm in diameter at a relatively time as compared to tumors formed in AREG WT PyMT mice. Histologically, AREG KO tumors appear more differentiated and display a greater proportion of tumor stroma. This prompted us to further hypothesize that AREG may have diverse effects in the tumoral or stromal compartment of developing tumors. To individually assess the effects of AREG knockout in the tumor cells and the stroma, we utilized a transplantation model in which we prepared tumor chunks from AREG WT and KO PyMT tumors and transplanted them into AREG WT and KO mice. Through this model, we have found that AREG expression in the tumor has a greater impact on tumor growth and progression than the tumor stroma. Regardless of the AREG status of the recipient mice, AREG KO tumors display a significant growth impairment and reduced transplant efficiency. In summary, these results demonstrate that AREG is important for mammary tumor progression. Citation Format: Serena P. Chiang, George S. Karagiannis, John S. Condeelis, Jeffrey E. Segall. Investigating the role of amphiregulin in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2017-1844

Abstract 1974: Novel function of PCNA in mammary tumor development and distant metastasis through tyrosine phosphorylation

Abstract Breast cancer is the most common cancer besides skin cancer and is the second leading cause of cancer-related death in women in the US. Despite intensive study, metastasis and tumor dormancy continue to be major hurdles to eradicate this disease. PCNA forms a homotrimer ring encircling the DNA double helix and acts as a sliding platform indispensable for DNA replication, damage repair, and chromatin remodeling. While a positive role of PCNA in is conceivable, how PCNA functions in tumor progression remains elusive due to the embryonic lethal phenotype in PCNA-deficient mice. We have shown that PCNA is regulated by phosphorylation at tyrosine 211 (Y211) meditated through multiple growth factor-stimulated signaling pathways. Y211 phosphorylation enhances chromatin-bound PCNA to promote cell proliferation and is frequently detected in multiple human cancers including breast cancer. To further dissect its biological functions, we have generated the PCNA211F/211F “knock-in” mice in which Y211 is replaced with a structurally similar but non-phosphorylatable amino acid phenylalanine (F). Comparing to wild-type mice, PCNA211F/211F mice have significantly reduced mammary tumor formation upon induction by the carcinogen DMBA. To understand its biological role in oncogene-induced mammary tumorigenesis, the PCNA211F/211F mice were crossed with the MMTV-PyMT mice in which mammary gland expression of the oncogene polyoma virus middle T antigen (PyMT) is driven by the mouse mammary tumor virus (MMTV) enhancer/promoter. PCNA211F/211F;MMTV-PyMT mice have statistically significant but only moderate decrease in mammary tumor development. However, their lung metastasis is dramatically ablated compared to PCNAWT/WT;MMTV-PyMT mice harboring primary tumors of similar size. These results suggest that pY211-PCNA has an essential function not only in tumorigenesis but more importantly in progression to distant metastasis. Consistently, mammary tumors derived from PCNAWT/WT;MMTV-PyMT mice exhibits more pronounced stromal activity than tumors of PCNA211F/211F;MMTV-PyMT mice. The suppression of metastasis and stromal activity in PCNA211F/211F;MMTV-PyMT mice is associated with reduced number of tumor-initiating cells (TICs) as determined by in vitro and in vivo analyses. Further study using syngeneic orthotopic model of transplanting murine cancer cells into the mammary glands of PCNAWT/WT and PCNA211F/211F mice unveils an unexpected function of pY211-PCNA in the stromal compartment on tumor development. Thus, our study reveals a novel tumor-promoting function of PCNA in both parenchymal and stromal compartments besides its traditional role in cell proliferation. Given our prior findings that the signaling pathways leading to Y211 phosphorylation can also be inhibited by FDA-approved drugs, our results suggest that pY211-PCNA may be a new druggable mechanism to target tumor metastasis. Citation Format: Yuan-Liang Wang, Lanie Chang, Susan E. Waltz, Shao-Chun Wang. Novel function of PCNA in mammary tumor development and distant metastasis through tyrosine phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2017-1974

Abstract 5757: Study of human mammary tumor virus (HMTV) in human breast cancer by NanoString nCounter and FISH analysis

Abstract Human Mammary Tumor Virus (HMTV), a retrovirus 90-98% homologous to Mouse Mammary tumor Virus (MMTV), the etiological agent of murine mammary cancer, has been detected by PCR in 40% of American women’s breast cancers, but not in healthy tissues of the same breast (Cancer Research 1995, 55:5173-79; Clin. Cancer Research 2001, 7-283-4). A complete 9.9 kb HMTV proviral sequence has been detected in breast cancer genomic DNA (Cancer Res 2001,61:1754-9). HMTV viral particles with betaretrovirus characteristics have been isolated from metastatic breast cancer cells in effusions (MSSM cells). Expression of HMTV proteins has been detected in MSSM cells by western blot, fluorescence-activated cell sorting analysis, and immunofluorescence assays but not in normal mammary epithelial cells (J Virol Methods 2010, 163(1): 157-61). PCR is a sensitive technique susceptible of contamination, which due to amplification, could result in false positive detection. Although murine DNA was not found by PCR, the possibility still exists that HMTV in human DNA could be a result of laboratory contamination. Despite the substantial evidence supporting the presence of HMTV in breast cancer the controversy continues because the chance of contamination has led to doubt that murine-like viruses are human pathogens. We now report additional analyses of breast cancer specimens employing other methods for HMTV detection that reinforce previous findings. NanoString nCounter, a new technology using specific 100 mer oligonucleotides as probes bound to reporter molecules that can detect HMTV gag, env and LTR sequences as well as two murine specific probes in a single reaction without amplification. Flourescence in situ hybridization (FISH) assays with a HMTV probe is a technology that permits visualization of HMTV proviral DNA in the nuclei of surgical samples of breast cancer as well as in MSSM cells. Results from Nanostring nCounter, and FISH analysis show the presence of HMTV DNA in surgical cancer specimens and in MSSM cells. The data exclude contamination and confirm the authenticity of HMTV in human breast cancer. Next-generation sequencing and other studies are in progress to determine the role of HMTV in the pathogenesis of breast carcinoma. Citation Format: Stella M. Melana, Joseph Tripodi, Digna Nosike, Jaffer Shabnam, Polly Etkind, Beatriz GT Pogo, James F. Holland. Study of human mammary tumor virus (HMTV) in human breast cancer by NanoString nCounter and FISH analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5757. doi:10.1158/1538-7445.AM2017-5757

Bromodomain inhibition shows antitumoral activity in mice and human luminal breast cancer.

BET bromodomain inhibitors, which have an antitumoral effect against various solid cancer tumor types, have not been studied in detail in luminal breast cancer, despite the prevalence of this subtype of mammary malignancy. Here we demonstrate that the BET bromodomain inhibitor JQ1 exerts growth-inhibitory activity in human luminal breast cancer cell lines associated with a depletion of the C-MYC oncogene, but does not alter the expression levels of the BRD4 bromodomain protein. Interestingly, expression microarray analyses indicate that, upon JQ1 administration, the antitumoral phenotype also involves downregulation of relevant breast cancer oncogenes such as the Breast Carcinoma-Amplified Sequence 1 (BCAS1) and the PDZ Domain-Containing 1 (PDZK1). We have also applied these in vitro findings in an in vivo model by studying a transgenic mouse model representing the luminal B subtype of breast cancer, the MMTV-PyMT, in which the mouse mammary tumor virus promoter is used to drive the expression of the polyoma virus middle T-antigen to the mammary gland. We have observed that the use of the BET bromodomain inhibitor for the treatment of established breast neoplasms developed in the MMTV-PyMT model shows antitumor potential. Most importantly, if JQ1 is given before the expected time of tumor detection in the MMTV-PyMT mice, it retards the onset of the disease and increases the survival of these animals. Thus, our findings indicate that the use of bromodomain inhibitors is of great potential in the treatment of luminal breast cancer and merits further investigation.

Mouse mammary tumour virus (MMTV) and human breast cancer with neuroendocrine differentiation

BackgroundMouse mammary tumour viruses (MMTVs) may have a role in a subset of human breast cancers. MMTV positive human breast cancers have similar histological characteristics to neuroendocrine breast cancers and to MMTV positive mouse mammary tumours. The purpose of this study was to investigate the expression of neuroendocrine biomarkers – synaptophysin and chromogranin, to determine if these histological characteristics and biomarker expression were due to the influences of MMTV.MethodsImmunohistochemistry analyses to identify synaptophysin and chromogranin were conducted on a series of human breast cancers in which (i) MMTV had been previously identified and had similar histological characteristics to MMTV positive mouse mammary tumours and (ii) MMTV positive mouse mammary tumours.ResultsThe expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumors were all positive (7 of 7 specimens – 100% positive). The expression of synaptophysin and chromogranin in MMTV positive human breast cancers was much less prevalent (3 of 22 – 14%). There was no expression of synaptophysin and chromogranin in the normal breast tissue control specimens.DiscussionIt is not possible to draw any firm conclusions from these observations. However, despite the small numbers of MMTV positive mouse mammary tumours in this study, the universal expression in these specimens of synaptophysin and chromogranin proteins is striking. This pattern of synaptophysin and chromogranin expression is very different from their expression in MMTV positive human breast cancers. The reason for these differences is not known.ConclusionsThe high prevalence of positive expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and low expression of synaptophysin and chromogranin in MMTV positive human breast cancers indicates that MMTV is not usually associated with neuroendocrine human breast cancers.

The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure.

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation.

Molecular Signaling of Progesterone, Growth Hormone, Wnt, and HER in Mammary Glands of Dogs, Rodents, and Humans: New Treatment Target Identification.

Mammary tumors are the most common form of neoplasia in the bitch. Female dogs are protected when they are spayed before the first estrus cycle, but this effect readily disappears and is already absent when dogs are spayed after the second heat. As the ovaries are removed during spaying, ovarian steroids are assumed to play an essential role in tumor development. The sensitivity toward tumor development is already present during early life, which may be caused by early mutations in stem cells during the first estrus cycles. Later on in life, tumors arise that are mostly steroid-receptor positive, although a small subset of tumors overexpressing human epidermal growth factor 2 (HER2) and some lacking estrogen receptor, progesterone receptor (PR), and HER2 (triple negative) are present, as is the situation in humans. Progesterone (P4), acting through PR, is the major steroid involved in outgrowth of mammary tissue. PRs are expressed in two forms, the progesterone receptor A (PRA) and progesterone receptor B (PRB) isoforms derived from splice variants from a single gene. The dog and the whole family of canids have only a functional PRA isoform, whereas the PRB isoform, if expressed at all, is devoid of intrinsic biological activity. In human breast cancer, overexpression of the PRA isoform is related to more aggressive carcinomas making the dog a unique model to study PRA-related mammary cancer. Administration of P4 to adult dogs results in local mammary expression of growth hormone (GH) and wing less-type mouse mammary tumor virus integration site family 4 (Wnt4). Both proteins play a role in activation of mammary stem cells. In this review, we summarize what is known on P4, GH, and Wnt signaling in canine mammary cancer, how the family of HER receptors could interact with this signaling, and what this means for comparative and translational oncological aspects of human breast cancer development.