Search

Background Despite extensive molecular epidemiological studies, the prevalence and characteristics of Mouse Mammary Tumor Virus-Like Virus (MMTV-LV) in Chinese women breast cancer are still unclear. Besides, the prevalence of MMTV-LV in women breast cancer tissue varies in different countries and its dependent factors remain inconclusive. Methods In the first part of the study, a case-control study was performed. 119 breast cancer samples (84 from Northern China and 35 from Southern China) and 50 breast fibroadenoma specimens were collected from Chinese women patients. MMTV-like env sequence and the homology to MMTV env gene were analysed by semi-nested polymerase chain reaction (PCR). We also explored the association of MMTV-LV prevalence with sample sources (Southern and Northern China) and patients’ clinicopathological characteristics. To investigate the dependent factors of the prevalence of MMTV-LV in breast cancer worldwide, a meta-analysis was conducted in the second part of the study. Results We found that the prevalence of MMTV-LV was much higher in breast cancer tissues (17.65%) than that in breast fibroadenoma specimens (4.00%) (P P P Conclusion The distribution of house mice may be a crucial environmental factor that explains the geographic differences in human breast cancer incidence. Our findings may provide a potential avenue of prevention, diagnosis and treatment for breast cancer.

The expression of Merlin tumor suppressor protein encoded by Neurofibromin 2 (NF2) gene is remarkably decreased in metastatic breast cancer tissues. In order to recapitulate clinical evidence, we generated a unique, conditional Nf2‐knockout (Nf2−/−) mouse mammary tumor model. Merlin‐deficient breast tumor cells and Nf2−/− mouse embryonic fibroblasts (MEFs) displayed a robustly invasive phenotype. Moreover, Nf2−/− MEFs presented with notable alterations in redox management networks, implicating a role for Merlin in redox homeostasis. This programmatic alteration resonated with pathways that emerged from breast tumor cells engineered for Merlin deficiency. Further investigations revealed that NF2‐silenced cells supported reduced activity of the Nuclear factor, erythroid 2 like 2 antioxidant transcription factor, concomitant with elevated expression of NADPH oxidase enzymes. Importantly, mammary‐specific Nf2−/− in an Mouse mammary tumor virus Neu + murine breast cancer model demonstrated accelerated mammary carcinogenesis in vivo. Tumor‐derived primary organoids and cell lines were characteristically invasive with evidence of a dysregulated cellular redox management system. As such, Merlin deficiency programmatically influences redox imbalance that orchestrates malignant attributes of mammary/breast cancer., Merlin‐deficient cells have an under‐functioning Nuclear factor, erythroid 2 like 2 antioxidant system, as well as a NOX‐enabled redox production system in hyperdrive. This leads to intensified oxidative stress that converges upon unrestricted cell growth and intensification of malignant attributes. Thus, Merlin deficiency disables the cellular redox management system. The findings uncover a novel mechanism by which Merlin intersects with redox homeostasis in restricting malignant characteristics.

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of inter-human dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer.

Significance Human breast cancer develops after a long period of latency under premalignant conditions. Strategies to target the premalignant conditions have yet to materialize since the molecular mechanisms remain obscure. Here, we discovered that FRS2β, expressed in a subset of mammary epithelial cells, directly activates nuclear factor–κB (NF-κB) and drives the initiation and promotion of the stroma-rich premalignant conditions. The FRS2β-triggered activation of NF-κB takes place in the early endosomes, the organelles, which have not been believed to be a major place for NF-κB signaling. The endosome signaling should be a novel focus for targeting therapy for prevention of breast cancer. This work paves a new way to develop preventive strategies of breast tumor development., Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2β, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2β deficiency in mouse mammary tumor virus (MMTV)–ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2β-deficient premalignant tissues. We found that colocalization of FRS2β and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor–κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB–induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2β contain more stroma. The elucidation of the FRS2β–NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

Background Breast cancer, although the most frequently diagnosed malignant tumor in humans, has a less clear etiology compared to other frequent cancer types. Mouse-mammary tumor virus (MMTV) is involved in breast cancer in mice and dogs and might play a role in the etiology of some breast cancers in humans, since it has been identified in 20-40% of breast cancer samples in Western Europe, USA, Australia and some other parts of the world’s population. The purpose of our study was to identify MMTV DNA sequences in breast tissue samples from breast cancer patients who underwent curative surgery in our regional center in Romania, EU. MethodsWe selected 75 patients with non-metastatic breast cancer treated surgically with curative intent, which did not undergo any neoadjuvant treatment. Out of these patients, 50 underwent radical lumpectomy and 25 modified radical mastectomy. We searched using PCR the MMTV-like DNA env sequence in the breast cancer tissue and normal breast tissue obtained from the same patients. ResultsNone of the examined samples was positive for MMTV-like target sequences on PCR.ConclusionsWe could not prove that MMTV plays a role in the etiology of breast cancer in our patient group. This finding is similar to publications of other geographically related research groups and might be due to the fact that only the Mus musculus domesticus mouse species was proven to carry infectious MMTV, but not the Mus musculus musculus species, which is specific to South-Eastern Europe (including Romania) and some parts of Asia.

Simple Summary Rlip knockout has been reported to prevent cancer in highly cancer-susceptible mice lacking p53, and Rlip knockdown kills many types of cancer cells. In humans, breast cancer shows diverse characteristics, including HER2-driven subtypes and viral-driven subtypes. HER2 can be targeted; however, escape of the cancer from targeted therapies remains a problem. In this work we evaluated the capacity of Rlip knockout to prevent breast cancer in genetically engineered mouse models of HER2-driven breast cancer (Erbb2 model) and polyomavirus-driven breast cancer (PyVT model). We found that in Erbb2 mice, Rlip knockout significantly delayed oncogenesis and reduced the expression of genes associated with poor prognosis in patients. In PyVT mice, Rlip knockout did not delay oncogenesis or tumor growth, but Rlip knockdown reduced tumor metastasis to the lung. We conclude that Rlip inhibitors may significantly improve survival in HER2-positive patients, but are unlikely to offer benefits to patients with polyomavirus-associated tumors. Abstract We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Samah A Loutfy,1 Zeinab F Abdallah,1 Mohamed Shaalan,2 Mohamed Moneer,3 Adel Karam,3 Manar M Moneer,4 Ibrahim M Sayed,5,6 Amer Ali Abd El-Hafeez,7,8 Pradipta Ghosh,8– 10 Abdel-Rahman N Zekri1 1Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt; 2Surgical Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt; 3Surgical Oncology Department, Materia Teaching Hospital, Cairo, Egypt; 4Biostatistics and Epidemiology Department, National Cancer Institute, Cairo University, Cairo, Egypt; 5Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt; 6Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, CA, USA; 7Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt; 8Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, 92093, USA; 9Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA; 10Moores Cancer Center, University of California, San Diego, La Jolla, CA, 92093, USACorrespondence: Samah A LoutfyVirology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Fom El-Khalig, Cairo, 11796, EgyptTel +201222840964Email samaly183@yahoo.comBackground: Mouse mammary tumor virus (MMTV) is thought to have a role in human breast cancer (BC) pathogenesis. BRCA1 and 2 genes mutations are well-established risk factors for BC. The purpose of this study was to evaluate the presence of MMTV in familial and non-familial Egyptian breast cancer patients. We also aimed to establish a correlation between BRCAs genes mutations and MMTV infection in those patients.Patients and Methods: The study was included 80 BC patients and 10 healthy women were included as a control group. We used PCR to amplify a 250-bp MMTV-like env sequence. We also used PCR followed by direct sequencing to identify the genetic variation of exons 2, 13, 19 of BRCA1 gene and exon 9 and region f of exon 11 of BRCA2 gene. High resolution melting (HRM) analysis was used to screen the selected exons of BRCA1/2 genes in order to detect different variants.Results: MMTV DNA-like env sequences were detected in 70%, 76% of familial and non-familial BC patients, respectively, and it was not detected in any of the control subjects. The presence of viral sequences was associated with larger tumor size in the sporadic patients. Seventy BC patients showed variations in BRCA1/2 genes according to HRM analysis and sequencing analysis showed two different sequences of polymorphism among 22 familial and non-familial BC patients.Conclusion: MMTV DNA was present among BC patients and it was associated with increased tumor growth. This indicates a potential role for MMTV in BC patients with and without deleterious mutation in BRCA1/2 genes.Keywords: breast cancer, MMTV, BRCA1/2, HRM, Egypt

Background Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. Methods We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. Results The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. Conclusions Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Background Breast cancer is a complex disease which is defined by an intrinsic heterogeneity at the histopathological and molecular levels, as well as in response to therapy. It remains the second leading cause of cancer death among women worldwide despite advances in screening, detection and new therapeutic options. Therefore, it is important to establish relevant preclinical mouse models to study new therapeutics and tumor biology. Genetically engineered mouse models (GEMMs) have been developed in order to understand the molecular, biochemical and cellular functions of oncogenes or tumor suppressor genes. However, the application of GEMMs is constrained due to the spontaneous nature of tumor onset and progression and high cost of breeding. Homograft tumor models, which are derived from and retain the histopathological and molecular features of GEMMs, can be used as faithful surrogates of human tumors. Methods We generated a series of homograft tumor models from GEMMs overexpressing human epidermal growth factor receptor 2 (HER2, also known as ERBB2) or polyomavirus middle T antigen (PyMT) driven by the mouse mammary tumor virus (MMTV) promoter, or Simian Virus 40 T-antigen (SV40 Tag) under the promotion of the rat prostate steroid binding protein (C3(1)), which are commonly used GEMMs in preclinical breast cancer research.1 2 Models were generated by transplanting the mammary tumors into donor animals. Furthermore, we characterized the homograft tumors through histopathological analysis, immunohistochemical analysis, and immune profiling, as well as immunotherapeutic, cytotoxic and targeted therapy. Results Nine breast cancer homograft models were developed from MMTV-ERBB2, MMTV-PyMT and C3(1)-Tag GEMMs, including six hormone receptor negative and HER2 positive models (mBR9013, mBR9026, mBR9027, mBR9028, mBR9029, mBR9030), one hormone receptor positive and HER2 negative model (mBR6174) and two triple negative models (mBR6004, mBR9014). Immune profiling of six models showed enriched macrophage infiltration in the tumor microenvironment. Immunotherapy treatment with anti-mPD-1 and anti-mCTLA-4 produced tumor growth inhibition (TGI) of 98% and 110%, respectively, in the triple negative model mBR9014, accompanied by tumor regression. HER2 targeted treatment with lapatinib produced robust response with TGI ranging from 48% to 97% in one HER2 negative and two HER2 positive models. Varying response to the cytotoxic treatments was observed among different models, with cisplatin producing robust response of TGI over 80% in all five of the tested models. Conclusions We have generated and characterized a series of mouse breast cancer homograft models from GEMMs to facilitate both mechanistic investigation and preclinical testing of novel therapeutics. Ethics Approval Animal experiments were conducted in accordance with animal welfare law, approved by local authorities, and in accordance with the ethical guidelines of CrownBio (Taicang). References Sakamoto K, Schmidt JW, Wagner K-U. Mouse models of breast cancer. Methods Mol Biol 2015;1267:47–71. Usary J, Darr DB, Pfefferle AD, et al. Overview of genetically engineered mouse models of distinct breast cancer subtypes. Curr Protoc Pharmacol 2016;72:14.38.1–14.38.11.

The Tientsin Albino 2 (TA2) mouse has a high incidence of spontaneous breast cancer (SBC) in the absence of external inducers or carcinogens. The initiation of SBC is related to mouse mammary tumor virus (MMTV) infection and pregnancy. Pathologic analysis showed that breast cancer cells in TA2 mice are triple negative. Our previous study confirmed that fibroblast growth factor receptor 2 (FGFR2) expression increased in SBC tissue compared to that in their corresponding normal breast tissues of TA2 mice. The present study focused on the function of the FGFR2/STAT3 signaling pathway in the initiation of SBC. In this study, the expression of FGF3, FGFR2, STAT3, p-STAT3Tyr705, and p-STAT3Ser727 was detected in serum and normal mammary gland tissues of TA2 mice with different number of pregnancies and SBC. The proliferation, invasiveness, and migration abilities of MA-891 cells from TA2 SBC were compared before and after cryptotanshinone and Stattic treatment. Transient siRNA transfection was used to detect the invasiveness, and migration abilities to avoid the off-targets effects. Downstream protein expression of STAT3 was also detected in MA-891 cells and TA2 xenografts from MA-891 inoculation. In addition, STAT3 expression was analyzed in 139 cases of human breast cancer including 117 cases of non-triple negative breast cancer (non-TNBC) (group I) and 22 cases of triple-negative breast cancer (TNBC) (group II). Results of our study confirmed that MMTV-LTR amplification, and FGFR2, p-STAT3Tyr705, p-STAT3Ser727 expression increased with the number of pregnancies in the breast tissue of TA2 mice and were the highest in SBC. Serum FGF3 expression of SBC was higher than it of TA2 mice with different number of pregnancies. After STAT3 was inhibited, the abilities of proliferation, invasiveness, and migration in MA-891 decreased and the expression levels of STAT3, p-STAT3Ser727, p-STAT3Tyr705, Bcl2, cyclin D1, and c-myc in MA-891 and animal xenografts were also down-regulated. In human breast cancer, STAT3 expression was significantly higher in TNBC than that in non-TNBC. Our results showed that the FGFR2/STAT3 signaling pathway may be related to SBC initiation in TA2 mice. Inhibition of STAT3 can decrease proliferation, invasiveness, and migration in MA-891 cells and the growth of TA2 xenografts.

Canonical WNT/β- catenin signaling plays pivotal roles in mammary development and tumorigenesis; and aberrant activation of this pathway is frequently observed in human breast cancer, correlating with poor outcome. However, the mechanisms underlying WNT-mediated mammary tumorigenesis remain incompletely understood. Here, we used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop aggressive mammary adenomas, to examine whether Limb-Bud-and-Heart (LBH) - a WNT/β-catenin target transcription co-factor overexpressed in human triple-negative breast cancers with WNT pathway hyperactivation, contributes to WNT-induced tumorigenesis. We found LBH is specifically overexpressed in basal epithelial tumor cells of MMTV-WNT1 mammary tumors reminiscent of its’ basal cell-restricted expression pattern in the normal postnatal mammary gland. To determine the role of LBH in mammary tumorigenesis, we crossed MMTV-Wnt1 mice with epithelial-specific Keratin 14/K14-Cre;Lbh(loxP) knockout mice. Mammary glands from virgin LBH-deficient MMTV-Wnt1 mice exhibited reduced hyperplasia, cell proliferation and increased apoptosis. Importantly, LBH inactivation in mammary epithelium significantly delayed tumor onset in MMTV-Wnt1 transgenic mice, with a median tumor-free survival of 32.5 weeks compared to 22.5 weeks in control LBH wild type MMTV-Wnt1 mice (p

SUMMARY MED1 (mediator subunit 1)co-amplifies with HER2, but its role in HER2-driven mammary tumorigenesis is still unknown. Here, we generate MED1 mammary-specific overexpression mice and cross them with mouse mammary tumor virus (MMTV)-HER2 mice. We observe significantly promoted onset, growth, metastasis, and multiplicity of HER2 tumors by MED1 overexpression. Further studies reveal critical roles for MED1 in epithelial-mesenchymal transition, cancer stem cell formation, and response to anti-HER2 therapy. Mechanistically, RNA sequencing (RNA-seq) transcriptome analyses and clinical sample correlation studies identify Jab1, a component of the COP9 signalosome complex, as the key direct target gene of MED1 contributing to these processes. Further studies reveal that Jab1 can also reciprocally regulate the stability and transcriptional activity of MED1. Together, our findings support a functional cooperation between these co-amplified genes in HER2+ mammary tumorigenesis and their potential usage as therapeutic targets for the treatment of HER2+ breast cancers., Graphical Abstract, In brief In this study, Yang et al. generate a more clinically relevant MMTV-HER2/MMTV-MED1 mammary tumor mouse model and discover the critical roles and molecular mechanisms of MED1 overexpression in mediating the aggressive phenotypes of HER2+ tumor progression, metastasis, cancer stem cell formation, and therapy resistance.

Breast cancer is a heterogeneous disease, and stratification of patients is fundamental to the success of treatment modalities. Breast tumors deficient in BRCA1 are mostly associated with basal-like breast cancers and targeted therapeutics for this disease subtype are still lacking. In order to address whether macroautophagy/autophagy inhibition will be effective in BRCA1-deficient mammary tumors, we generated mice with conditional deletion of an essential autophagy gene, Rb1cc1, along with Brca1 and Trp53, through utilization of the K14-Cre transgene. We found that Rb1cc1 deletion suppressed tumorigenesis in the BRCA1-deficient model when compared to wild type and heterozygous Rb1cc1 controls. However, in contrast to previous studies in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) model, tumor growth and the distribution of histological subtypes were not affected by loss of RB1CC1. Interestingly, loss of RB1CC1 decreased mitochondrial mass and oxidative respiratory capacity of these tumor cells, along with a decrease in the phosphorylation of MTOR substrates and transcript levels of genes involved in mitochondrial biogenesis. Importantly, we observed an increased sensitivity to mitochondrial disrupting agents upon loss of RB1CC1. Consequently, our data showed that combination of an autophagy inhibitor, spautin-1, along with a mitochondrial complex I inhibitor, metformin, was more effective in limiting oxidative respiratory capacity, colony-forming ability and tumor growth. Altogether, our results indicate that inhibition of autophagy can increase the benefits of metformin treatment in BRCA1-deficient breast cancers.

The human betaretrovirus and the closely related mouse mammary tumor virus have been linked with the development of cholangitis and mitochondrial antibody production in patients with primary biliary cholangitis (PBC) and mouse models of autoimmune biliary disease, respectively. In vitro, betaretroviruses have been found to stimulate the expression of mitochondrial autoantigens on the cell surface of biliary epithelial cells. In vivo, both mitochondrial autoantigens and viral proteins have been shown to be co-expressed in biliary epithelium and lymphoid tissue. Notably, both mice and humans make poor antibody responses to betaretrovirus infection, whereas proinflammatory responses to viral proteins have been observed in T lymphocyte studies. Furthermore, proviral integration studies have confirmed the presence of human betaretrovirus in biliary epithelium of patients with PBC. Preliminary proof of principal studies using combination antiretroviral therapy have shown that suppression of viral expression is associated with sustained biochemical response. As the previous regimen used was poorly tolerated, further randomized controlled trials are planned to determine whether betaretrovirus infection plays an important role in the development of PBC.

The initiation of spontaneous breast cancer (SBC) in Tientsin Albino 2 (TA2) mice is related to mouse mammary tumor virus (MMTV) infection, and MMTV amplification is hormonally regulated. To explore the insertion site of MMTVLTR in TA2 mouse genome, reverse PCR and nested PCR were used to amplify the unknown sequence on both sides of the MMTV-LTRSAG gene in SBC and normal breast tissue of TA2 mice. Furthermore, the clinicopathological significance of the insertion site was evaluated in 43 samples of normal breast tissue, 46 samples of breast cystic hyperplasia, 54 samples of ductal carcinoma in situ, 142 samples of primary breast cancer and 47 samples of lymph node metastatic breast cancer by RNA in situ hybridization. We confirmed that the insertion site of the MMTV-LTRSAG gene was located between Igκv2-112 and Igκv14-111 in chromosome 6 of TA2 mouse. IGκC was localized in the stromal cells of TA2 mouse with SBC and in human breast cancer tissues. Tumor cells were negative for IGκC in RNA in situ hybridization. The positive staining index of IGκC in stromal cells was the highest in lymph node metastatic breast cancer, followed by primary breast cancer, ductal carcinoma in situ, and breast cystic hyperplasia. Furthermore, the positive staining index of IGκC was related to the expression of ER, PR, HER2 and Ki-67. Our findings showed that stromal IGκC expression was associated with the initiation of SBC in TA2 mice. IGκC may be a high-risk factor for the initiation and progression of human breast cancer.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac–treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

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.

Background In 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. Methods Using 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. Results We 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. Conclusions These findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis. Electronic supplementary material The online version of this article (10.1186/s12885-017-3817-0) contains supplementary material, which is available to authorized users.

// Monica K. VanKlompenberg 1, 2, 5 , Emily Leyden 2, 3 , Anne H. Arnason 2, 3 , Jian-Ting Zhang 4 , Casey D. Stefanski 2, 3 and Jenifer R. Prosperi 1, 2, 3 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine–South Bend, South Bend, IN, USA 2 Harper Cancer Research Institute, South Bend, IN, USA 3 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA 4 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA 5 Current address: Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA Correspondence to: Jenifer R. Prosperi, email: jrprospe@iupui.edu Keywords: Adenomatous Polyposis Coli; breast cancer; STAT3; chemoresistance; doxorubicin Received: September 07, 2017 Accepted: October 16, 2017 Published: November 01, 2017 ABSTRACT 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;Apc Min/+ 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;Apc Min/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;Apc Min/+ 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.

Mouse 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.

Background Multiple 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. Methods We 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. Results Standard 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. Conclusions These 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.

Myeloid-derived suppressor cells (MDSCs) are innate immune cells that acquire the capacity to suppress adaptive immune responses during cancer. It remains elusive how MDSCs differ from their normal myeloid counterparts, which limits our ability to specifically detect and therapeutically target MDSCs during cancer. Here, we used single-cell RNAseq to compare MDSC-containing splenic myeloid cells from breast tumor-bearing mice to wildtype controls. Our computational analysis of 14,646 single-cell transcriptomes reveals that MDSCs emerge through a previously unrealized aberrant neutrophil maturation trajectory in the spleen giving rise to a unique chemokine-responsive, immunosuppressive cell state that strongly differs from normal myeloid cells. We establish the first MDSC-specific gene signature and identify novel surface markers for improved detection and enrichment of MDSCs in murine and human samples. Our study provides the first single-cell transcriptional map defining the development of MDSCs, which will ultimately enable us to specifically target these cells in cancer patients.One Sentence SummaryWe used single cell transcriptomics to identify the unique molecular features distinguishing myeloid-derived suppressor cells (MDSCs) from their normal, myeloid counterparts, which enabled us to reveal distinct transitory gene expression changes during their maturation in the spleen, and to identify novel cell surface markers for improved detection and isolation of MDSCs.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that plays a causal role in the development of breast cancer and lymphoma in mice. Closely related sequences that share 91–99% nucleotide identity with MMTV have been repeatedly found in humans with neoplastic and inflammatory diseases. Evidence for infection with a betaretrovirus has been found in patients with breast cancer and primary biliary cholangitis and referred to as the human mammary tumor virus and the human betaretrovirus (HBRV), respectively. Using the gold standard technique of demonstrating retroviral infection, HBRV proviral integrations have been detected in cholangiocytes, lymph nodes, and liver of patients with primary biliary cholangitis. However, the scientific biomedical community has not embraced the hypothesis that MMTV like betaretroviruses may infect humans because reports of viral detection have been inconsistent and robust diagnostic assays are lacking. Specifically, prior serological assays using MMTV proteins have produced divergent results in human disease. Accordingly, a partial HBRV surface (Su) construct was transfected into HEK293 to create an ELISA. The secreted HBRV gp52 Su protein was then used to screen for serological responses in patients with breast cancer and liver disease. A greater proportion of breast cancer patients (n = 98) were found to have serological reactivity to HBRV Su as compared to age- and sex-matched control subjects (10.2% versus 2.0%, P=0.017, OR = 5.6 [1.25–26.3]). Similarly, the frequency of HBRV Su reactivity was higher in patients with primary biliary cholangitis (n = 156) as compared to blood donors (11.5% vs. 3.1%, P=0.0024, OR = 4.09 [1.66–10.1]). While the sensitivity of the HBRV Su ELISA was limited, the assay was highly specific for serologic detection in patients with breast cancer or primary biliary cholangitis, respectively (98.0% [93.1%–99.7%] and 97.0% [93.4%–98.6%]). Additional assays will be required to link immune response to betaretrovirus infection and either breast cancer or primary biliary cholangitis.

We tested how adjuvants effect in a cancer vaccine model using an epitope derived from an autoactivation loop of membrane-type protease serine protease 14 (PRSS14; loop metavaccine) in mouse mammary tumor virus (MMTV)-polyoma middle tumor-antigen (PyMT) system and in 2 other orthotopic mouse systems. Earlier, we reported that loop metavaccine effectively prevented progression and metastasis regardless of adjuvant types and TH types of hosts in tail-vein injection systems. However, the loop metavaccine with Freund's complete adjuvant (CFA) reduced cancer progression and metastasis while that with alum, to our surprise, were adversely affected in 3 tumor bearing mouse models. The amounts of loop peptide specific antibodies inversely correlated with tumor burden and metastasis, meanwhile both TH1 and TH2 isotypes were present regardless of host type and adjuvant. Tumor infiltrating myeloid cells such as eosinophil, monocyte, and neutrophil were asymmetrically distributed among 2 adjuvant groups with loop metavaccine. Systemic expression profiling using the lymph nodes of the differentially immunized MMTV-PyMT mouse revealed that adjuvant types, as well as loop metavaccine can change the immune signatures. Specifically, loop metavaccine itself induces TH2 and TH17 responses but reduces TH1 and Treg responses regardless of adjuvant type, whereas CFA but not alum increased follicular TH response. Among the myeloid signatures, eosinophil was most distinct between CFA and alum. Survival analysis of breast cancer patients showed that eosinophil chemokines can be useful prognostic factors in PRSS14 positive patients. Based on these observations, we concluded that multiple immune parameters are to be considered when applying a vaccine strategy to cancer patients.

Diabetes and cancer share multiple risk factors including obesity and insulin resistance, but the potential relationship between these two major diseases is unknown. Here we examined the metabolic effects of high-fat diet (HFD) feeding in female MMTV-PyMT mice, a breast cancer model expressing the polyoma middle T antigen driven by the Mouse Mammary Tumor Virus promoter. Female PyMT and wild type (WT) mice were fed a HFD (55% fat by calories) for 4 weeks starting at 4∼5 weeks of age (n=10-12/group). During HFD, PyMT mice became more obese than WT mice with significant increases in both fat and lean mass (Figure 1; *P In conclusion, these results demonstrate that tumor-bearing female PyMT mice developed increased obesity and insulin resistance after high-fat feeding, and our findings implicate an important relationship between obesity, cancer, and type 2 diabetes. Disclosure H. Noh: None. H. Kang: None. J. Mercado-Matos: None. R.H. Friedline: None. S. Suk: None. X. Hu: None. D.A. Tran: None. L.A. Tauer: None. M. Ko: None. J. Choi: None. T. Luong: None. Y. Tsuchida: None. L.M. Shaw: None. J.K. Kim: None. Funding National Institutes of Health (5U2C-DK093000)

The WASF3 gene has been implicated in cancer cell movement, invasion, and metastasis by regulating genetic pathways important in these processes. Invasion and metastasis assays, however, are largely centered on xenograft models in immune-compromised mice. To facilitate analysis of the role of WASF3 in the spontaneous development of cancer cell metastasis, we generated a Wasf3 null strain by deleting exons 4 and 5, which encode essential motifs for Wasf3 function. On exposure to cre-recombinase a stop codon is generated immediately downstream in exon 6. Using a cytomegalovirus (CMV)-cre strain, Wasf3 constitutively was inactivated, which led to viable mice with no visible morphologic or behavioral abnormalities. There was no abnormal development or function of the mouse mammary gland in the Wasf3 null mice and brain development was normal. In the mouse mammary tumor virus (MMTV)-driven polyoma middle-T oncogene strain, which shows early onset breast cancer development and metastasis, Wiskott-Aldrich syndrome protein family member 3 (Wasf3) is up-regulated in metastatic lesions. When this oncogene was introduced onto the Wasf3-null background, although metastasis was observed in these mice, there was a reduction in the number and size of metastatic lesions in the lungs. These data provide evidence for a role in WASF3 in the development of metastasis in a spontaneous model of breast cancer.

Development and progression of breast cancer is an outcome of strong interplay between proto-oncogenes as well as environmental factors. Among proto-oncogenes, c-myc, a multifunctional transcription factor (TF), is one of the most highlighted one, whereas among environmental factors Mouse Mammary Tumor Virus (MMTV)-like virus is a widely discussed agent. Both, c-myc and MMTV-like virus, are known to individually correlate with the poor prognosis of breast cancer. However, no study has ever been reported to determine their mutual association in breast cancer patients. In this study, our aim was to quantify and compare c-myc mRNA in MMTV-like virus-positive and virus-negative-histopathological types of breast cancer. At first, biopsy samples of 105 breast cancer patients with known histopathological types were collected and screened for the presence of MMTV-like virus. To quantify mRNA level of c-myc, quantitative-Polymerase Chain Reaction (qPCR) was used. Next, c-myc expression was compared in MMTV-like virus-positive and virus-negative-histopathological types as of breast cancer. Statistical analysis was done using GraphPad Prism 7 Software. Molecular analysis revealed that 69 (65.72%) out of 105 samples were positive for MMTV-like virus. Moreover, invasive types of breast cancer exhibited increased (3-13 folds higher) expression of c-myc as compared to baseline representing normal control comprising of 15 tumor-free biopsy samples of breast cancer patients. Whereas, non-invasive types of breast cancer showed only 1-3 folds increase in the expression of c-myc as compared to normal control. Furthermore, virus-positive and virus-negative samples had different levels of c-myc mRNA. Positive status of MMTV-like virus was noticed to significantly associate with c-myc expression increasing it from 1.87-folds in virus-negative patient samples to 4.31-folds in virus-positive patient samples (p-value

Purpose: Tientsin Albino 2 (TA2) mice have a high incidence of spontaneous breast cancer. Tumor initiation is related to mouse mammary tumor virus (MMTV) infection. MMTV is hormonally regulated and may promote tumor formation via Wnt/β-catenin signaling pathway. This study attempts to clarify the relationship between β-catenin expression and the initiation and metastasis of spontaneous breast cancer in TA2 mice. Materials and Methods: Pathological samples illustrating the development of spontaneous breast cancer in TA2 mice were collected and the presence of virus particles was verified in the cancer tissue by electron microscope. Expression of Wnt/β-catenin signaling-pathway-related proteins including β-catenin, Wnt 5a, GSK-3β, and cyclin D1 were detected. MA-891 cell line derived from TA2 spontaneous breast cancer was cultured and siRNA was used to inhibit the expression of β-catenin in the primary culture cell line. Cell cycle analyses and comparisons of the invasiveness and migration capability of tumor cells were performed before and after β-catenin inhibition. Downstream protein expression of β-catenin was studied by western blot, co-immunoprecipitation assay. Tumorigenesis and metastasis were compared with that of negative control, siRNA control, and siRNA β-catenin-1512. Furthermore, proteins related to the proliferation and invasion of tumor were detected by western blot. Results: β-catenin expression was found to be located in the membrane and cytoplasm in normal mammary tissue and precancerous lesions, respectively. However, in the breast cancer tissue, β-catenin expression was located in the nuclei. After transfection with siRNA-1512, the cells showed decreased proliferation, invasiveness and migration capability, tumorigenicity, and metastasis, and the expression of the proteins related to tumor proliferation and metastasis such as c-myc, Cyclin D1, MMP-9, and VEGF were down-regulated. Conclusion: These results confirmed that the expression and location of β-catenin were associated with the initiation and metastasis of spontaneous breast cancer in TA2 mice.

World widely, Breast cancer is the most common type of cancer amongst women. External and internal factors both are responsible for beginning, advancement and development of breast cancer and from many studies it is well known that environmental elements have a significant effect in etiology of breast cancer. When it comes to environmental factors, effects of viruses are of great concern. Virus-associated cancer brings up a cancer in which the malicious conversion of the host’s diseased cells results due to any viral infection. Until now it has learned that human papillomaviruses (HPV), Epstein–Barr (EBV) and mouse mammary tumor virus (MMTV) are the prime candidate viruses that act as agents of causing breast cancer. The initiator is only one in a sequence of steps essential for malignant cells development. Breast cancer could be a hereditary disease but it is expected to account for a very small percentage of breast cancer cases. From this idea it is obvious that there should be more efforts required in evaluating the role of viruses in carcinogenesis that could be characterized by further confounding and synergistic special effects of carcinogenic aspects still exact role of viruses in tumorigenesis is unclear, but it seem like that these are responsible for causing the disease. Link: http://aeirc-edu.com/ojs14/index.php/IJEHSR/article/view/127/335

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer‐related death globally. R‐spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation‐related protein 53 (Trp53). Moreover, the Hippo/yes‐associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/β‐catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap‐dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2‐induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt‐driven tumors of the liver.

The inheritance of mutated suppressor genes, such as BRCA1 and BRCA2, is acknowledged as an etiological factor in hereditary breast carcinoma (HBC). Two different molecular mechanisms are possible; the Knudson’s “two hits” or the gene haploinsufficiency. Etiology of sporadic breast carcinoma (SBC) is not known, although data support the possible role of the betaretrovirus Mouse Mammary Tumor Virus (MMTV). This study analyzes the presence of MMTV exogenous sequences in two representative groups of HBC and SBC, excluding any contamination by murine and retroviral material and endogenous betaretroviruses. The 30.3% of 56 SBC contained MMTV sequences, against the 4.2% of 47 HBC (p < 0.001). Cases positive for viral sequences showed the presence of p14, signal peptide of the MMTV envelope precursor. This result was expected based on the fact that HBCs, having a specific genetic etiology, do not need the action of a carcinogenetic viral agent. Moreover, the striking results obtained by comparing two groups of vastly different tumors represent an additional element of quality control: the distinction between HBC and SBC is so well-defined that results cannot be ascribed to mere coincidence. This paper strengthens the hypothesis for a viral etiology for human sporadic breast carcinoma.

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.

Presenting Author: Loutfy SA Introduction: Oncogenic viruses including Mouse Mammary Tumor Virus (MMTV), Adenovirus (ADV), Polyomavirus (PoV), Human Papillomavirus (HPV), and Epstein–Barr virus (EBV) are one of the leading probable direct causes of Breast cancer (BC) worldwide. According to the National population-based registry program in Egypt (2008-2011) accounting for 32% of all female cancers. The present study aimed at evaluating the presence of these five oncogenic viruses among Egyptian familial and non-familial BC patients and their impact on the clinical course of the disease. Patients & Methods: The current study was conducted on 20 cases of BC patients without treatment and 10 healthy women as a control, aged between 18-45 years. Fresh frozen tissue and blood were obtained from BC patients, whereas only blood was obtained from control. All samples were subjected to qualitative and quantitative PCR assays to measure viral loads. The results were correlated to the clinicopathological parameters of breast cancer disease. Results: MMTV, ADV, PoV, HPV and EBV DNAs have been detected in 11 (55%),13 (65 %),8 (40%), 0 (0%) and 0 (0%) respectively, of 20 blood BC patients using qualitative PCR. PCR assay. Upon screening of 13 fresh frozen tissues, MMTV, ADV, PoV, HPV and EBV DNAs were present in 12(92%), 12(92%), 2(15.3%), 11(84.6%), and 11 (84.6) respectively of BC patients using quantitative PCR assay. Regarding viral load, ADV and HPV DNAs appeared to be the most replicative viruses out of all tested oncogenic viruses where their load ranged between 40 and 130,000 copies/ml. None of the tested viruses was detected in the control group. The presence of DNA of the 5 tested viruses was not associated with any clinicopathological parameters except larger tumor size was more common among BC patients with positive MMTV results. Conclusion: MMTV and ADV were the most common viruses present in tissue and blood of BC patients, tissue specimens are better for viral detection especially for EBV and papillomaviruses. The presence of oncogenic viral infection among young BC females could be considered as one of the risk external factor and could affect the path of BC disease and its prognosis.

The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.

Purpose The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. Methods Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1–11 years later developed into breast cancer. Results MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. Discussion These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. Conclusion Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Breast cancer is the most common cancer among women worldwide. Roles of the Epstein-Barr, Merkel cell polyoma and mouse mammary tumor viruses in breast carcinogenesis are still controversial although any relationship would clearly be important for breast cancer etiology, early detection and prevention. In the present study associations between EBV, MMTV and Merkel cell polyoma virus and breast cancer in 100 Iranian patients were evaluated using paraffin-embedded tissues. EBER RNA and expression of p53 and large T antigen were evaluated by real time PCR and CD34, p63, HER2, PR and ER markers were studied by immunohistochemistry. EBV was detected in 8/100 (8%), MMTV in 12/100 (12%), MPy in 3/100 (3%) and EBER RNA in 18/100 (18%) cases. None of the control samples demonstrated any of the viruses. p53 was suppressed in EBV, MPy and MMTV positive samples. The large T antigen rate was raised in MPy positive samples. Our results showed that EBV, MMTV and the Merkel cell polyoma virus are foundwith some proportion of breast cancers in our patients, suggesting that these viruses might have a significant role in breast cancer in Kerman, southeast of Iran.

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. The possible mechanism behind these polyglutamine-induced alterations in AR function is discussed., Highlights • Spinal bulbular muscular atrophy is caused by a polyQ expanded androgen receptor. • Function of AR with expanded polyQ tract was analyzed in Xenopus oocytes. • AR with expanded polyQ tract has increased DNA binding but reduced gene activation.

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.

Purpose We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could promote the development of preinvasive and invasive breast cancer in mouse mammary tumor virus (MMTV-erbB2) mice with estrogen receptor-positive tumors. Methods MMTV-erbB2 mice were randomly divided into three experimental groups with 20 mice in each group. MMTV-erbB2 mice were treated with daily subcutaneous injections of vehicle or rhG-CSF (low-rhG-CSF group, rhG-CSF 0.125 µg; vehicle-rhG-CSF group, normal saline 0.25 µg; and high-rhG-CSF group, rhG-CSF 0.25 µg) at 3 months of age. Cellular and molecular mechanisms of G-CSF action in mammary glands were investigated via immunohistochemistry and reverse transcription polymerase chain reaction. Results Low, but not high, rhG-CSF doses significantly accelerated mammary tumorigenesis in MMTV-erbB2 mice. Short-term treatment with rhG-CSF could significantly promote the development of preinvasive mammary lesions. The cancer prevention effect was associated with reduced expression of proliferating cell nuclear antigen, cluster of differentiation 34, and signal transducers and activators of transcription 3 in mammary glands by >80%. Conclusion We found that G-CSF was regulated by rhG-CSF both in vitro and in vivo. Identification of G-CSF genes helped us further understand the mechanism by which G-CSF promotes cancer. Low doses of rhG-CSF could significantly increase tumor latency and increase tumor multiplicity and burden. Moreover, rhG-CSF effectively promotes development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.

The wingless-type mouse mammary tumor virus integration site family (Wnt) pathway plays a major role in the carcinogenesis of colorectal cancer (CRC). Its most important effector, the nuclear β-catenin, influences not only transcription but also the proliferation and dedifferentiation of the colonic mucosa. This induces an epithelial-mesenchymal transition which ultimately can lead to the development of cancer and the formation of metastases. However, little is known about the exact interaction and context-sensitive expression of Wnt-pathway effectors in the primary tumor and corresponding metastasis. Therefore, this study assessed the expression of the three most important effectors of the Wnt pathway, β-catenin, adenomatous polyposis coli (APC) and Wnt-1, in the primary tumor and corresponding metastasis of patients with CRC. Immunohistochemical staining of β-catenin, APC and Wnt-1 was performed in paraffin-embedded tissue samples of the primary tumor, and the corresponding hepatic and nodal metastasis samples from 24 patients with metastatic CRC. Isotype antibodies were used as negative controls. The results were visualized using the ABC-method. Analysis of the primary tumor comprised of a separate evaluation of the central tumor area as well as the invasion front. There was a significant overexpression of nuclear β-catenin at the tumor invasion front (P

Bone is one of the most common sites of cancer metastasis in humans and is a significant source of morbidity and mortality. Bone metastases are considered incurable and result in pain, pathologic fracture, and decreased quality of life. Animal models of skeletal metastases are essential to improve the understanding of the molecular pathways of cancer metastasis and growth in bone and to develop new therapies to inhibit and prevent bone metastases. The ideal animal model should be clinically relevant, reproducible, and representative of human disease. Currently, an ideal model does not exist; however, understanding the strengths and weaknesses of the available models will lead to proper study design and successful cancer research. This review provides an overview of the current in vivo animal models used in the study of skeletal metastases or local tumor invasion into bone and focuses on mammary and prostate cancer, lymphoma, multiple myeloma, head and neck squamous cell carcinoma, and miscellaneous tumors that metastasize to bone.

The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV)–Neu] and a triple-negative/basal-like [C3(1)–Simian virus 40 large T antigen (Tag)] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9), an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1)-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1), an MMP9 substrate, were increased in C3(1)-Tag;Mmp9−/− compared to C3(1)-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1)-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

A significant improvement in agonist activity of the previously described 2-aryloctahydrophenanthrene-2,3,7-triol series of dissociated glucocorticoid receptor agonists (DAGRs) was achieved by modifying the substitution at C3 from (S)-3-hydroxy to (R)-3-hydroxy-3-methyl. The IC50 of the prototype 13 in the efficacy assay measuring repression of IL-1 induced MMP-13 expression was 3.5 nM, exhibiting 87% of the maximal effect of dexamethasone (DEX). It displayed a dissociated profile by exhibiting 42% of the maximal effect of DEX in a mouse mammary tumor virus (MMTV) luciferase reporter transactivation assay. Compound 13 and analogues containing heterocyclic replacements for the C2 phenyl and modified B rings showed high repression of TNFα production in human whole blood, with IC50 values (43-167 nM) approaching the level of DEX (21 nM). On the basis of X-ray structures and force field calculations, the overall potency of this series was attributed to a favorable conformation of the C2α phenyl, induced by the neighboring C3α methyl.

The wingless-type mouse mammary tumor virus integration site family (WNT) signaling pathway is involved in wound healing and fibrosis. We evaluated the WNT signaling pathway in peritoneal membrane injury. We assessed WNT1 protein expression in the peritoneal effluents of 54 stable peritoneal dialysis (PD) patients and WNT-related gene expression in ex vivo mesothelial cell cultures from 21 PD patients. In a transforming growth factor-β (TGF-β)-mediated animal model of peritoneal fibrosis, we evaluated regulation of the WNT pathway and the effect of WNT inhibition on peritoneal fibrosis and angiogenesis. WNT1 and WNT2 gene expression were positively correlated with peritoneal membrane solute transport in PD patients. In the mouse peritoneum, TGF-β-induced peritoneal fibrosis was associated with increased expression of WNT2 and WNT4. Peritoneal β-catenin protein was significantly upregulated after infection with adenovirus expressing TGF-β (AdTGF-β) along with elements of the WNT signaling pathway. Treatment with a β-catenin inhibitor (ICG-001) in mice with AdTGF-β-induced peritoneal fibrosis resulted in attenuation of peritoneal angiogenesis and reduced vascular endothelial growth factor. Similar results were also observed with the WNT antagonist Dickkopf-related protein (DKK)-1. In addition to this, DKK-1 blocked epithelial-mesenchymal transition and increased levels of the cell adhesion protein E-cadherin. We provide evidence that WNT signaling is active in the setting of experimental peritoneal fibrosis and WNT1 correlates with patient peritoneal membrane solute transport in PD patients. Intervention in this pathway is a possible therapy for peritoneal membrane injury.

There are a number of publications suggesting that a virus highly related to mouse mammary tumor virus (MMTV) is involved in human breast cancer but this is not universally accepted. A recent paper concludes that MMTV is probably not involved in human breast cancer, based primarily on PCR data, because less than one copy of MMTV proviral DNA per cell can be found in human breast cancer samples. However, this conclusion assumes the tumor arose from a single cisactivation event due to insertional mutagenesis. Here we outline other mechanisms by which MMTV and other related retroviruses cause tumorigenesis. Since insertional mutagenesis is not the only mechanism by which retroviruses can induce tumorigenesis, we suggest that it is not yet possible to rule out a role for a virus highly related to mouse mammary tumor virus (MMTV) in human breast cancer.

Fluorescence-activated cell sorting (FACS) is a common method to identify and to isolate subpopulations within a complex mixture of cells based on their light scatter and fluorescent staining profiles. FACS is widely used to enrich for normal tissue and tumor cells that have stem cell potential. Whereas FACS protocols using conventional breast cancer cell lines are relatively routine, additional technical challenges are encountered when sorting for cell populations from freshly digested solid tumors, particularly for use in downstream cancer stem cell (CSC) assays. First, it is more difficult to isolate live, single cells from whole tumors, and second, single tumor cells prepared from enzymatically digested tumors are typically more sensitive to cell death following the physical stresses of digestion, pipetting, and sorting. Herein methods are described that have been optimized to harvest and to FACS profile viable tumor epithelial cells digested from late-stage mammary tumors originating in the mouse mammary tumor virus (MMTV)-polyomavirus middle T antigen (PyMT) transgenic mouse. Protocols were designed to enrich for single, viable, MMTV-PyMT tumor cell populations sorted by FACS and to facilitate the collection of sorted cell subpopulations suitable for head-to-head comparison of CSC activity by tumorsphere assays in vitro or limiting dilution transplantation in vivo.

Enteric viruses encounter a multitude of environments as they traverse the gastrointestinal tract. The interaction of enteric eukaryotic viruses with members of the host microbiota impacts the outcome of infection. Infection with several enteric viruses is impaired in the absence of the gut microbiota, specifically bacteria. The effects of bacteria on virus biology are diverse. Poliovirus capsid stability and receptor engagement are positively impacted by bacteria and bacterial lipopolysaccharides. Norovirus utilizes histo-blood group antigens produced by enteric bacteria to attach and productively infect B cells. Lipopolysaccharides on the envelope of mouse mammary tumor virus promote a tolerogenic environment that allows for the establishment of viral persistence. Reovirus binds Gram negative and Gram-positive bacteria through bacterial envelope components to enhance virion thermostability. Through the direct engagement of bacteria and bacterial components, viruses evolved diverse ways to impact the outcome of infection.