USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of function impacts MMTV promoter activity.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)-driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.

Decoding the mystery of MMTV-like virus and its relationship with breast cancer metastasis.

BACKGROUND: MMTV causes mammary tumors in mice, and it is associated with invasive and aggressive forms of breast cancer in humans. However, the underlying mechanisms are yet unknown. We aimed to determine the MMTV-like virus (MMTV-LV) association with histological types of breast cancer, nodal involvement, and metastasis. METHODS: First, 105 breast cancer biopsies and 15 disease-free biopsies were collected. Details of clinicopathological characteristics were retrieved from patients' records. The status of MMTV-LV was already known for these biopsy samples. Associations of MMTV-LV prevalence with LNM status and metastatic history were determined. Next, quantitative PCR (qPCR) was used to quantify env gene mRNA in biopsies positive for MMTV-LV. Expression of the env gene was compared against different histopathological types of mammary tumor, LNM status, and metastasis by performing Ordinary One Way ANOVA followed by Tukey's multiple comparisons test. RESULTS: MMTV-LV prevalence was found to have no significant association with LNM or metastatic history. As compared to normal control, expression of the env gene was significantly higher (>2.8 folds) in invasive samples (P-value: < 0.01). Expression was also higher (3.28 and 2.89 folds) in patient samples with LNM (P-value: 0.0006) or metastatic history (P-value: < 0.0001), respectively. CONCLUSION: We conclude that MMTV-LV prevalence is not associated with LNM status or breast cancer metastasis; samples with invasive phenotypes, nodal involvement, and metastasis exhibit significantly higher expression of the MMTV-like env gene.

Interferon-gamma plasma levels and presence of mouse mammary tumor virus-like env gene: Implications on the pathogenesis of breast cancer.

Mouse mammary tumor virus (MMTV) is a retrovirus that has been associated with the development of breast cancer (BC) in mice. The identification of a 95% homologous gene sequence to MMTV in human BC samples has increased interest in this hypothesis. This virus in humans received the name of mouse mammary tumor virus-like (MMTV-like). Several cytokines may be involved in the interactions between MMTV and the immune system, such as interferon-gamma (IFN-γ), which can enhance Th1-mediated antitumor immune response but it can also play a protumorigenic role by transmitting antiapoptotic and proliferative signals. Little is known about the antiviral immune response in a microenvironment with the presence of MMTV-like in BC patients. Therefore, the purpose of the present study was to quantify the plasma levels of IFN-γ in the peripheral blood of 123 neoplasia-free donors and 98 BC patients of different molecular subtypes, by enzyme-linked immunosorbent assay (ELISA), and evaluate the association of these plasma levels with the detection of the MMTV-like env gene in tumor tissue. Correlation analyzes involving IFN-γ plasma levels and clinical-pathological parameters were performed by Kendall Tau-c test. In our study, a decrease in IFN-γ levels was observed in the group of BC patients (30.85 ± 57.49 pg/ml) compared to the control group (115.00 ± 176.80 pg/ml) (p < 0.0001). In the analysis by stratified BC molecular subtypes, Luminal-A (30.79 ± 61.04 pg/ml; p < 0.0001), Luminal-B (24.74 ± 25.78 pg/ml; p = 0.0188) and triple-negative (23.95 ± 40.45 pg/ml; p = 0.0005) had a lower plasma level compared to control group. There was no significant difference between IFN-γ plasma levels of MMTV-like DNA positive samples compared to MMTV-negative samples (p = 0.2056). In general BC, patients with larger tumor size had higher IFN-γ plasma levels (Tau-c = 0.202; p = 0.019). By analyzing the MMTV-like env negative samples, we could identify that IFN-γ plasma levels were higher in larger tumor size (Tau-c = 0.222; p = 0.020) and with greater lymph node involvement (Tau-c = 0.258; p = 0.042). Also, higher IFN-γ plasma levels were observed in patients with higher histopathological grades (Tau-c = 0.384; p = 0.019) in MMTV-like env positive samples. For the first time, we assessed the association between plasma levels of IFN-γ and the presence of the MMTV-like env gene in BC samples. However, more studies are needed to clarify whether the high levels of IFN-γ in MMTV-like env positive samples are reflecting a possible antiviral immune response or whether this cytokine is promoting tumor growth.

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice.

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

The Role of a Betaretrovirus in Human Breast Cancer: Enveloping a Conundrum.

Most of the evidence that a human betaretrovirus (HBRV/HMTV) highly related to mouse mammary tumour virus (MMTV) has an etiological role in breast cancer has been summarized in a recent comprehensive Special Issue of "Viruses" entitled "Human Betaretrovirus (HBRV) and Related Diseases". Shortly after publication of this special issue, a detailed analysis of aligned env sequences was published and concluded that (i) MMTV and HBRV/HMTV cannot be distinguished on the basis of aligned env sequences and (ii) more sequence data covering the full-length env or HBRV/HMTV genomes from multiple isolates is needed. Although productive infection of human cells by MMTV (and presumably HBRV/HMTV) has been shown, it is imperative that the receptor(s) enabling HBRV/HMTV to infect human cells are defined. Moreover, there is currently no compelling data for common integration sites, in contrast to MMTV induced mammary tumorigenesis in mice, suggesting that other mechanisms of tumorigenesis are associated with HBRV/HMTV infection. These issues need to be resolved before a clear link between MMTV/HBRV/HMTV and human breast cancer can be concluded.

Life after Cleavage: The Story of a ß-Retroviral (MMTV) Signal Peptide-From Murine Lymphoma to Human Breast Cancer.

An increasing body of evidence in recent years supports an association of the betaretrovirus mouse mammary tumor virus (MMTV) with human breast cancer. This is an issue that still raises heated controversy. We have come to address this association using the signal peptide p14 of the MMTV envelope precursor protein as a key element of our strategy. In addition to its signal peptide function, p14 has some significant post endoplasmic reticulum (ER)-targeting characteristics: (1) it localizes to nucleoli where it binds key proteins (RPL5 and B23) involved (among other activities) in the regulation of nucleolar stress response, ribosome biogenesis and p53 stabilization; (2) p14 is a nuclear export factor; (3) it is expressed on the cell surface of infected cells, and as such, is amenable to, and successfully used, in preventive vaccination against experimental tumors that harbor MMTV; (4) the growth of such tumors is impaired in vivo using a combination of monoclonal anti-p14 antibodies or adoptive T-cell transfer treatments; (5) p14 is a phospho-protein endogenously phosphorylated by two different serine kinases. The phosphorylation status of the two sites determines whether p14 will function in an oncogenic or tumor-suppressing capacity; (6) transcriptional activation of genes (RPL5, ErbB4) correlates with the oncogenic potential of MMTV; (7) finally, polyclonal anti-p14 antibodies have been applied in immune histochemistry analyses of breast cancer cases using formalin fixed paraffin-embedded sections, supporting the associations of MMTV with the disease. Taken together, the above findings constitute a road map towards the diagnosis and possible prevention and treatment of MMTV-associated breast cancer.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.

The Viral Origin of Human Breast Cancer: From the Mouse Mammary Tumor Virus (MMTV) to the Human Betaretrovirus (HBRV).

A Human Betaretrovirus (HBRV) has been identified in humans, dating as far back as about 4500 years ago, with a high probability of it being acquired by our species around 10,000 years ago, following a species jump from mice to humans. HBRV is the human homolog of the MMTV (mouse mammary tumor virus), which is the etiological agent of murine mammary tumors. The hypothesis of a HMTV (human mammary tumor virus) was proposed about 50 years ago, and has acquired a solid scientific basis during the last 30 years, with the demonstration of a robust link with breast cancer and with PBC, primary biliary cholangitis. This article summarizes most of what is known about MMTV/HMTV/HBRV since the discovery of MMTV at the beginning of last century, to make evident both the quantity and the quality of the research supporting the existence of HBRV and its pathogenic role. Here, it is sufficient to mention that scientific evidence includes that viral sequences have been identified in breast-cancer samples in a worldwide distribution, that the complete proviral genome has been cloned from breast cancer and patients with PBC, and that saliva contains HBRV, as a possible route of inter-human infection. Controversies that have arisen concerning results obtained from human tissues, many of them outdated by new scientific evidence, are critically discussed and confuted.

Isolation of a Human Betaretrovirus from Patients with Primary Biliary Cholangitis.

A human betaretrovirus (HBRV) has been linked with the autoimmune liver disease, primary biliary cholangitis (PBC), and various cancers, including breast cancer and lymphoma. HBRV is closely related to the mouse mammary tumor virus, and represents the only exogenous betaretrovirus characterized in humans to date. Evidence of infection in patients with PBC has been demonstrated through the identification of proviral integration sites in lymphoid tissue, the major reservoir of infection, as well as biliary epithelium, which is the site of the disease process. Accordingly, we tested the hypothesis that patients with PBC harbor a transmissible betaretrovirus by co-cultivation of PBC patients' lymph node homogenates with the HS578T breast cancer line. Because of the low level of HBRV replication, betaretrovirus producing cells were subcloned to optimize viral isolation and production. Evidence of infection was provided by electron microscopy, RT-PCR, in situ hybridization, cloning of the HBRV proviral genome and demonstration of more than 3400 integration sites. Further evidence of viral transmissibility was demonstrated by infection of biliary epithelial cells. While HBRV did not show a preference for integration proximal to specific genomic features, analyses of common insertion sites revealed evidence of integration proximal to cancer associated genes. These studies demonstrate the isolation of HBRV with features similar to mouse mammary tumor virus and confirm that patients with PBC display evidence of a transmissible viral infection.

Mouse Mammary Tumor Virus (MMTV) and MMTV-like Viruses: An In-depth Look at a Controversial Issue.

Since its discovery as a milk factor, mouse mammary tumor virus (MMTV) has been shown to cause mammary carcinoma and lymphoma in mice. MMTV infection depends upon a viral superantigen (sag)-induced immune response and exploits the immune system to establish infection in mammary epithelial cells when they actively divide. Simultaneously, it avoids immune responses, causing tumors through insertional mutagenesis and clonal expansion. Early studies identified antigens and sequences belonging to a virus homologous to MMTV in human samples. Several pieces of evidence fulfill a criterion for a possible causal role for the MMTV-like virus in human breast cancer (BC), though the controversy about whether this virus was linked to BC has raged for over 40 years in the literature. In this review, the most important issues related to MMTV, from its discovery to the present days, are retraced to fully explore such a controversial issue. Furthermore, the hypothesis of an MMTV-like virus raised the question of a potential zoonotic mouse-man transmission. Several studies investigate the role of an MMTV-like virus in companion animals, suggesting their possible role as mediators. Finally, the possibility of an MMTV-like virus as a cause of human BC opens a new era for prevention and therapy.

Incidence of HPVs, EBV, and MMTV-Like Virus in Breast Cancer in Qatar.

INTRODUCTION: Human papillomaviruses (HPVs), Epstein-Barr virus (EBV), and mouse mammary tumor virus-like virus (MMTV-like virus) can be present and contribute to breast cancer development and progression. However, the role of these oncoviruses and their crosstalk in breast cancer is still unclear. METHODS: We explored the co-presence of high-risk HPVs, EBV, and MMTV-like virus in 74 breast cancer samples from Qatar using PCR. RESULTS: We found the presence of HPV and EBV in 65% and 49% of our cancer sample cohorts; 47% of the samples are positive for both oncoviruses. The MMTV-like virus alone was detected in 15% of the samples with no significant association with clinicopathological features. The three oncoviruses were co-present in 14% of the cases; no significant association was noted between the co-presence of these viruses and the clinicopathological features. CONCLUSION: Despite the presence of the oncoviruses, additional studies are necessary to understand their interactions in human breast carcinogenesis.

Mouse Mammary Tumour Virus (MMTV) in Human Breast Cancer-The Value of Bradford Hill Criteria.

For many decades, the betaretrovirus, mouse mammary tumour virus (MMTV), has been a causal suspect for human breast cancer. In recent years, substantial new evidence has been developed. Based on this evidence, we hypothesise that MMTV has a causal role. We have used an extended version of the classic A. Bradford Hill causal criteria to assess the evidence. 1. Identification of MMTV in human breast cancers: The MMTV 9.9 kb genome in breast cancer cells has been identified. The MMTV genome in human breast cancer is up to 98% identical to MMTV in mice. 2. EPIDEMIOLOGY: The prevalence of MMTV positive human breast cancer is about 35 to 40% of breast cancers in Western countries and 15 to 20% in China and Japan. 3. Strength of the association between MMTV and human breast cancer: Consistency-MMTV env gene sequences are consistently five-fold higher in human breast cancer as compared to benign and normal breast controls. 4. Temporality (timing) of the association: MMTV has been identified in benign and normal breast tissues up to 10 years before the development of MMTV positive breast cancer in the same patient. 5. EXPOSURE: Exposure of humans to MMTV leads to development of MMTV positive human breast cancer. 6. Experimental evidence: MMTVs can infect human breast cells in culture; MMTV proteins are capable of malignantly transforming normal human breast epithelial cells; MMTV is a likely cause of biliary cirrhosis, which suggests a link between MMTV and the disease in humans. 7. Coherence-analogy: The life cycle and biology of MMTV in humans is almost the same as in experimental and feral mice. 8. MMTV Transmission: MMTV has been identified in human sputum and human milk. Cereals contaminated with mouse fecal material may transmit MMTV. These are potential means of transmission. 9. Biological plausibility: Retroviruses are the established cause of human cancers. Human T cell leukaemia virus type I (HTLV-1) causes adult T cell leukaemia, and human immunodeficiency virus infection (HIV) is associated with lymphoma and Kaposi sarcoma. 10. Oncogenic mechanisms: MMTV oncogenesis in humans probably differs from mice and may involve the enzyme APOBEC3B. CONCLUSION: In our view, the evidence is compelling that MMTV has a probable causal role in a subset of approximately 40% of human breast cancers.

Thermodynamics of unfolding mechanisms of mouse mammary tumor virus pseudoknot from a coarse-grained loop-entropy model.

Pseudoknotted RNA molecules play important biological roles that depend on their folded structure. To understand the underlying principles that determine their thermodynamics and folding/unfolding mechanisms, we carried out a study on a variant of the mouse mammary tumor virus pseudoknotted RNA (VPK), a widely studied model system for RNA pseudoknots. Our method is based on a coarse-grained discrete-state model and the algorithm of PK3D (pseudoknot structure predictor in three-dimensional space), with RNA loops explicitly constructed and their conformational entropic effects incorporated. Our loop entropy calculations are validated by accurately capturing previously measured melting temperatures of RNA hairpins with varying loop lengths. For each of the hairpins that constitutes the VPK, we identified alternative conformations that are more stable than the hairpin structures at low temperatures and predicted their populations at different temperatures. Our predictions were validated by thermodynamic experiments on these hairpins. We further computed the heat capacity profiles of VPK, which are in excellent agreement with available experimental data. Notably, our model provides detailed information on the unfolding mechanisms of pseudoknotted RNA. Analysis of the distribution of base-pairing probability of VPK reveals a cooperative unfolding mechanism instead of a simple sequential unfolding of first one stem and then the other. Specifically, we find a simultaneous "loosening" of both stems as the temperature is raised, whereby both stems become partially melted and co-exist during the unfolding process.

Revisiting the MMTV Zoonotic Hypothesis to Account for Geographic Variation in Breast Cancer Incidence.

Human breast cancer incidence varies by geographic location. More than 20 years ago, we proposed that zoonotic transmission of the mouse mammary tumor virus (MMTV) from the western European house mouse, Mus musculus domesticus, might account for the regional differences in breast cancer incidence. In the intervening years, several developments provide additional support for this hypothesis, including the limited impact of genetic factors for breast cancer susceptibility revealed by genome-wide association studies and the strong effect of antiretroviral therapy to reduce breast cancer incidence. At the same time, economic globalization has further expanded the distribution of M. m. domesticus to Asia, leading to a significant increase in breast cancer incidence in this region. Here, we revisit this evidence and provide an update to the MMTV zoonotic hypothesis for human breast cancer at a time when the world is recovering from the global COVID-19 zoonotic pandemic. We present evidence that mouse population outbreaks are correlated with spikes in breast cancer incidence in Australia and New Zealand and that globalization has increased the range of M. m. domesticus and MMTV. Given the success of global vaccination campaigns for HPV to eradicate cervical cancer, a similar strategy for MMTV may be warranted. Until breast cancer incidence is reduced by such an approach, zoonotic transmission of MMTV from mice to humans as an etiologic factor for breast cancer will remain controversial.

A Retrotranslocation Assay That Predicts Defective VCP/p97-Mediated Trafficking of a Retroviral Signal Peptide.

Studies of viral replication have provided critical insights into host processes, including protein trafficking and turnover. Mouse mammary tumor virus (MMTV) is a betaretrovirus that encodes a functional 98-amino-acid signal peptide (SP). MMTV SP is generated from both Rem and envelope precursor proteins by signal peptidase cleavage in the endoplasmic reticulum (ER) membrane. We previously showed that SP functions as a human immunodeficiency virus type 1 (HIV-1) Rev-like protein that is dependent on the AAA ATPase valosin-containing protein (VCP)/p97 to subvert ER-associated degradation (ERAD). SP contains a nuclear localization sequence (NLS)/nucleolar localization sequence (NoLS) within the N-terminal 45 amino acids. To directly determine the SP regions needed for membrane extraction and trafficking, we developed a quantitative retrotranslocation assay with biotin acceptor peptide (BAP)-tagged SP proteins. Use of alanine substitution mutants of BAP-tagged MMTV SP in retrotranslocation assays revealed that mutation of amino acids 57 and 58 (M57-58) interfered with ER membrane extraction, whereas adjacent mutations did not. The M57-58 mutant also showed reduced interaction with VCP/p97 in coimmunoprecipitation experiments. Using transfection and reporter assays to measure activity of BAP-tagged proteins, both M57-58 and an adjacent mutant (M59-61) were functionally defective compared to wild-type SP. Confocal microscopy revealed defects in SP nuclear trafficking and abnormal localization of both M57-58 and M59-61. Furthermore, purified glutathione S-transferase (GST)-tagged M57-58 and M59-61 demonstrated reduced ability to oligomerize compared to tagged wild-type SP. These experiments suggest that SP amino acids 57 and 58 are critical for VCP/p97 interaction and retrotranslocation, whereas residues 57 to 61 are critical for oligomerization and nuclear trafficking independent of the NLS/NoLS. Our results emphasize the complex host interactions with long signal peptides. IMPORTANCE Endoplasmic reticulum-associated degradation (ERAD) is a form of cellular protein quality control that is manipulated by viruses, including the betaretrovirus, mouse mammary tumor virus (MMTV). MMTV-encoded signal peptide (SP) has been shown to interact with an essential ERAD factor, VCP/p97 ATPase, to mediate its extraction from the ER membrane, also known as retrotranslocation, for RNA binding and nuclear function. In this paper, we developed a quantitative retrotranslocation assay that identified an SP substitution mutant, which is defective for VCP interaction as well as nuclear trafficking, oligomer formation, and function. An adjacent SP mutant was competent for retrotranslocation and VCP interaction but shared the other defects. Our results revealed the requirement for VCP during SP trafficking and the complex cellular pathways used by long signal peptides.

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

Dual recombinase action in the normal and neoplastic mammary gland epithelium.

We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.

Presence of high-risk HPVs, EBV, and MMTV in human triple-negative breast cancer.

Breast cancer, the most frequent disease amongst women worldwide, accounts for the highest cancer-related mortality rate. Triple-negative breast cancer (TNBC) subtype encompasses ~15% of all breast cancers and lack estrogen, progesterone, and HER2 receptors. Although risk factors for breast cancer are well-known, factors underpinning breast cancer onset and progression remain unknown. Recent studies suggest the plausible role of oncoviruses including human papillomaviruses (HPVs), Epstein-Barr virus (EBV), and mouse mammary tumor virus (MMTV) in breast cancer pathogenesis. However, the role of these oncoviruses in TNBC is still unclear. In the current study, we explored the status of high-risk HPVs, EBV, and MMTV in a well-defined TNBC cohort from Croatia in comparison to 16 normal/non TNBC samples (controls) using polymerase chain reaction assay. We found high-risk HPVs and EBV present in 37/70 (53%) and 25/70 (36%) of the cases, respectively. The most common HPV types are 52, 45, 31, 58 and 68. We found 16% of the samples positive for co-presence of high-risk HPVs and EBV. Moreover, our data revealed that 5/70 (7%) samples are positive for MMTV. In addition, only 2/70 (3%) samples had co-presence of HPVs, EBV, and MMTV without any significant association with the clinicopathological variables. While, 6/16 (37.5%) controls were positive for HPV (p = .4), EBV was absent in all controls (0/16, 0%) (p = .01). In addition, we did not find the co-presence of the oncoviruses in the controls (p > .05). Nevertheless, further investigations are essential to understand the underlying mechanisms of multiple-oncogenic viruses' interaction in breast carcinogenesis, especially TNBC.

A Milk-Fat Based Diet Increases Metastasis in the MMTV-PyMT Mouse Model of Breast Cancer.

A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.