MMTV Promoter Research Articles

Lysine-Cysteine-Serine-Tryptophan inserted into the DNA-binding domain of human mineralocorticoid receptor increases transcriptional activation by aldosterone

Due to alternative splicing in an ancestral DNA-binding domain (DBD) of the mineralocorticoid receptor (MR), humans contain two almost identical MR transcripts with either 984 amino acids (MR-984) or 988 amino acids (MR-988), in which their DBDs differ by only four amino acids, Lys,Cys,Ser,Trp (KCSW). Human MRs also contain mutations at two sites, codons 180 and 241, in the amino terminal domain (NTD). Together, there are five distinct full-length human MR genes in GenBank. Human MR-984, which was cloned in 1987, has been extensively studied. Human MR-988, cloned in 1995, contains KCSW in its DBD. Neither this human MR-988 nor the other human MR-988 genes have been studied for their response to aldosterone and other corticosteroids. Here, we report that transcriptional activation of human MR-988 by aldosterone is increased by about 50 % compared to activation of human MR-984 in HEK293 cells transfected with the TAT3 promoter, while the half-maximal response (EC50) is similar for aldosterone activation of MR-984 and MR-988. Transcriptional activation of human MR also depends on the amino acids at codons 180 and 241. Interestingly, in HEK293 cells transfected with the MMTV promoter, transcriptional activation by aldosterone of human MR-988 is similar to activation of human MR-984, indicating that the promoter has a role in the regulation of the response of human MR-988 to aldosterone. The physiological responses to aldosterone and other corticosteroids in humans with MR genes containing KCSW and with differences at codons 180 and 241 in the NTD warrant investigation.

Abstract 1452: A PIK3CA-driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies

Abstract Metaplastic Breast Carcinomas (MBC) are rare and aggressive breast cancers with mesenchymal or squamous components. While histologically heterogeneous, MBC are typically triple negative: they lack expression of ER, PR and HER2. Importantly, approximately 40% of MBC harbor oncogenic gain-of-function mutations in PIK3CA, the gene encoding for the catalytic subunit of PI3K. These alterations are targetable by an array of inhibitors of the PI3K-AKT-mTORC1 pathway. However, few animal models of MBC are available, and it is currently difficult to study MBC biology and response to therapy. Here, we describe the generation of a new mouse model of MBC driven by the PIK3CA H1047R mutation. We histologically and biologically characterize this model and assess potential therapeutic strategies. We used genetically-engineered mouse models to express the human PIK3CA H1047R mutation under the control of the MMTV promoter, which is mostly expressed in the mammary epithelium. Tumors developed in mice in about 1 year. We histologically characterized these tumors and generated cell lines, organoids, and tumor grafts in immunocompromised mice. The resulting model, termed MMTV-102, was evaluated for the response to PI3K-AKT-mTOR pathway inhibitors, both in vitro and in vivo. The MMTV-PIK3CAH1047R tumor was a high grade poorly differentiated MBC negative for ER, PR, and HER2. It presented squamous components, keratin depositions, mitotic figures, and vascular invasion and stained positive for the basal marker CK14. In addition, due to the presence of the PIK3CA H1047R mutation, it stained positive for phosphorylated AKT, PRAS40, and S6RP, indicating constitutive activation of the PI3K-AKT-mTORC1 pathway. MBC derived cell lines and organoids were confirmed to be triple negative and could form tumors in mice. In addition, these cellular models were sensitive to PI3Kα, pan-PI3K, and mTORC1 inhibitors in vitro and in vivo, but the mTORC1 inhibitor everolimus exhibited the strongest effect. When resistance to everolimus eventually developed in vivo, we established cell lines from these resistant tumors. In addition, we generated an everolimus resistant cell line following chronic drug exposure in vitro (MMTV-102 Par res). Both cellular models were tested for their sensitivity to the next-generation mTORC1 inhibitor RMC-5552 by proliferation and clonogenic assays. We show that our mouse model can originate MBC with an active PI3K pathway. These tumors are amenable to transplantation into immunocompromised mice and to generate cell lines and organoids, allowing us to develop a simple, versatile, and rapid model of MBC. Tumors derived from our model are sensitive to PI3Kα and mTORC1 inhibitors, but eventually develop resistance to these agents. The use of next-generation mTORC1 inhibitors can overcome resistance in some cases, providing a rationale for their implementation in the treatment of patients with MBC. Citation Format: Paola Roa, Valentina Foglizzo, Michelle R. de Marchena, Shraddha Chandthakuri, Emiliano Cocco, Pau Castel. A PIK3CA-driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1452.

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.

N-terminal domain influences steroid activation of the Atlantic sea lamprey corticoid receptor

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid and progesterone activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells that were transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells that were transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3. This indicated that the promoter has an important effect on NTD regulation of transcriptional activation of the CR by steroids. Our results also indicate that the entire lamprey CR sequence is needed for an accurate determination of steroid-mediated transcription.

Abstract 1348: LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models

Abstract Introduction: Cyclin E is an independent predictor of poor outcomes and response to treatment in breast cancer (BrCa). Expression of low-molecular-weight cyclin E (LMW-E) is associated with more aggressive disease in all BrCa subtypes. While tumor infiltrating lymphocytes (TILs) are more abundant in LMW-E+ tumors, high-TIL/LMW-E+ tumors have lower probability of pathological complete response (pCR) to neoadjuvant chemotherapy. We hypothesized that LMW-E induces immune changes that create a permissive microenvironment in the mammary gland for promoting tumor initiation and subsequent growth. We aimed to evaluate the role of mammary epithelial expression of LMW-E in the temporal induction of systemic and local immune responses that ultimately prime the mammary gland for tumor development. Methods: We generated a tri-transgenic mouse model capable of conditionally expressing human LMW-E under the control of the MMTV promoter in a p53 heterozygous background (MPT) upon doxycycline (Dox) administration. Female MPT mice were treated with Dox for 3, 6 and 9 months and age-matched untreated controls were sacrificed at each time point. An independent group of non-transgenic mice in a p53 heterozygous background were maintained +/- Dox as positive and negative controls. Mammary glands and peripheral organs (spleen, lung, bone marrow) were harvested for immune profiling by flow cytometry and multiplex immunofluorescence microscopy (mIF). Serum was also collected for cytokine/chemokine assessment. Immune profiling via flow cytometry was performed using two multi-color panels to assess basic immune and more specialized T-cell subsets. For the mIF experiments, two 5-marker panels were applied to mammary tissue. Results: Histological examination of MPT mammary glands over time showed a temporal increase in acinar proliferation and mitotic figures, which was further confirmed by the co-localization of panCK+ and Ki-67+ markers. We report that although immune cell frequencies changed with age, these specific changes were dependent on LMW-E induction as compared to the non-transgenic cohort of mice. LMW-E+ mammary glands showed a temporal enrichment in B cells, macrophages, T cells (CD4+, PD1+, CD4+Ki67+, and Tregs), cDCs, and panCK+, panCK+vimentin+ populations over time. By contrast, pDCs increased from 3 to 6 months but decreased in the pre-tumorigenic mammary gland of the 9-month old LMW-E+ mice. Conclusions: LMW-E induction mediates an increase in epithelial cell proliferation and epithelial-to-mesenchymal transition events that result in local immune alterations, specifically affecting T-cell subsets. Our findings suggest that the immunological changes driven by LMW-E lead to an immunosuppressive microenvironment that may promote tumor formation at the early stages of breast tumorigenesis. Citation Format: Sofia Mastoraki, Amriti R. Lulla, Sarah Schneider, Karen Clise-Dwyer, Morgan M. Green, Natalie W. Fowlkes, Kelly K. Hunt, Stephanie S. Watowich, Khandan Keyomarsi. LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1348.

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.

Abstract P1-03-09: The role of E2F5 in breast cancer progression and metastasis

Abstract Advances in cancer genomics and transcriptomics have provided a better understanding of the diverse genetic landscape driving breast cancer pathogenesis. Despite these advances, the molecular drivers of triple negative breast cancer, an aggressive and heterogenous disease, remains highly elusive. Utilizing a genomic approach, we examined genetic differences across mouse models of breast cancer and identified a gene that was consistently deleted in basal-like mouse models, the predominant intrinsic subtype of triple negative breast cancer. Specifically, we identified a consistent deletion in the transcription factor E2F5. In human breast cancer, E2F5 activity and expression is associated with better overall survival and relapse free survival. To further study the role of E2F5 in breast cancer, E2F5 was deleted in the mammary gland of FVB mice (E2F5cKO) through the use of a cre-lox system driven by the MMTV promoter. E2F5cKO mouse demonstrated a delay a mammary outgrowth, suggesting a potential role in mammary gland development. E2F5cKO mice were aged and observed for tumor development. This revealed mammary tumor formation after an extended latency of 19 months. The resulting mammary tumors demonstrated diverse histological patterns. E2F5cKO tumors also demonstrate high metastatic potential with 70% of tumor bearing mice developing metastasis to the lung. In addition, E2F5cKO tumors have a propensity to metastasize to axillary lymph nodes when transplanted into MMTV-Cre mice, a unique feature that is not commonly observed in other mouse models of breast cancer. To begin to understand the mechanism of tumor progression in E2F5cKO mice, differential gene expression was performed on gene expression data derived from E2F5 or GFP overexpression Human Mammary Epithelial Cells. The resulting differentially expressed genes were further filtered based on fold change, established role in cancer, potential E2F binding site and gene ontology. One of the leading candidates that arose from this analysis was KRas, an oncogene that is highly implicated in tumorigenesis. KRas expression negatively correlated with E2F5 expression. Using a bioinformatic approach, we demonstrated that this inverse relationship between KRas and E2F5 appears to be conserved in human breast cancer. Given this inverse relationship between E2F5 and KRas, we postulate that E2F5 deletion in the mammary gland of mice leads to increased KRas activity. Consistent with our hypothesis, western blot analysis revealed high activity in two major KRas signaling pathways, Akt and Erk, in the majority of E2F5cKO tumors. Together, this preliminary data suggest that E2F5 may behave as a tumor suppressor by negatively regulating the oncogene KRas. Citation Format: Briana To, Eran Robert Andrechek. The role of E2F5 in breast cancer progression and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-09.

SUN-001 Analysis of Isoform Specific Differential Splicing Effects in Prostate Cancer

The androgen receptor (AR) is a hormone activated transcription factor which is important for the growth and progression of prostate cancer (PCa). Metastatic PCa is treated with androgen deprivation therapy (ADT) based regimen but eventually, these tumours develop resistance through multiple mechanisms that reactivate AR. These include expression of constitutively active AR splice variants that lack a ligand binding domain such as AR-V7. To characterize the common and unique activities of AR and AR-V7, we engineered LNCaP and VCaP cell lines that express AR-V7 in response to doxycycline and performed RNA-Seq. Many genes were found to be regulated by both AR and AR-V7, but there were also genes regulated by only one isoform. Moreover, both isoforms altered mRNA splicing of numerous target genes, in some cases without altering overall levels of mRNA. In many cases, the alterations in splicing are AR isoform specific. To understand the differential splicing by AR isoforms, CD44 mini-gene splicing assay and studies of endogenous target genes are being done. The CD44 mini-gene contains an MMTV promoter and is a multi-functional molecule containing two variable exons, v4 and v5 that can be either included or excluded during the splicing process. Both AR and AR-V7 induce transcription from an MMTV promoter. Co-transfection of AR or AR-V7 with the CD44 mini-gene in Hela cells demonstrated that CD44 splicing was altered by AR and its variant AR-V7. R1881 treated samples showed a shift to RNA products resulting from the skipping of both variable exons. Treatment with R1881 showed a decrease in the PSI (Percent Spliced Inclusion) as compared to obtained in the absence of R1881 or AR-V7. In contrast, AR-V7 minimally induced single exon skipping as compared to vehicle. These observations suggest that AR and its variants can have marked effects on RNA splicing to produce radically different proteins with different splicing/inclusion ratio. Analysis of the RNA-Seq data revealed that multiple types of splicing events are affected by the AR isoforms. For instance, whether an isoform favours exclusion or inclusion of a particular exon is event specific. A comparison of splicing in VCaP and LNCaP models showed that only a subset of events were common to the two models. One of the most differentially regulated events in both lines is an exon inclusion/exclusion event in PGAP2. Both isoforms increase the inclusion/exclusion ratio. However, further analysis showed that AR-V7 preferentially increased the levels of inclusion, but did not affect the basal levels of skipping. Also, AR increased the inclusion/skipping ratio through repression of expression of the skipped product. Similar analyses for other splicing events are ongoing. Our studies will provide a better understanding for the molecular basis for isoform specific actions and the role of AR/AR-V7 in the regulation of mRNA transcription and splicing.

Transcriptional Activation by Glucocorticoid Receptor Studied by 3D Orbital Tracking Fluorescence Cross Correlation Spectroscopy

Gaining a deeper understanding of the genome and the interactions taking place on a molecular level in living cells is particularly difficult. But, attaining a better knowledge of the temporal dynamics of stochastic biomolecular processes and interactions is critical for understanding gene regulation and the process of transcription that in turn controls cellular metabolism. Through 3D orbital tracking we can visualize the transcription process by observing the temporal relationships between pre-mRNA transcription at an MMTV promoter the protein-DNA binding of the transcription factor glucocorticoid receptor (GR) in real-time by fluorescently labeling within a mammalian cell. This study establishes that we can effectively track these molecules of interest, fluorescently labeled pre-mRNA (labelled by PP7-GFP) and the transcription factor, GR (labelled by Halo-JF646) during the process of transcriptional activation (under induction by cortical steroid and Dexamethasone). This particular method allows for observation of single molecule and the determination of the temporal correlation between GR binding to the DNA and the activation of mRNA synthesis. The data collected improves our knowledge of how eukaryotic organisms regulate transcription and sheds light on previously unknown details about transcriptional activation and temporal coordination. This technique has the capacity to be expanded to the transcriptional processes of other genes, many of which could have weighty implications for our understanding of genome biology and human health and disease. 3D orbital tracking is opening new doors for further exploration into fundamental biochemical processes through a dynamic view of single fluorescent molecules within living systems at high speeds.

PO-290 Oncostatin M signalling mediates the crosstalk between cancer cells and tumour microenvironment and promotes breast cancer progression

IntroductionCytokines are important players in inflammation, a process highly associated with tumour initiation, tumour growth, angiogenesis and metastasis. Oncostatin M Receptor (OSMR) is a membrane receptor belonging to the interleukin 6 (IL6) receptor family and it has been shown to play a role in inflammation, angiogenesis and tumour progression. In breast cancer it has been associated with the EMT process, key component in cancer progression. While it is clear that cancer cells express the receptor OSMR it seems that the ligand Oncostatin M (OSM) is mainly secreted by the tumour stroma suggesting a possible existence of paracrine signalling between the tumour microenvironment and cancer cells. We previously showed that OSMR is frequently copy-number gained and over-expressed in squamous cell carcinoma, where it induces migration, invasion and metastasis. We now investigate the role of OSMR in breast cancer progression and its importance in mediating the communication with the tumour microenvironment.Material and methodsTo address this issue we used a wide array of tools including in vivo models and in vitro cell cultures of breast cancer cell lines together with co-cultures of stromal cells such as cancer-associated fibroblasts and macrophages. The role of OSMR in oncogenesis and metastasis was studied by generating a mouse line that expresses the PyMT oncogene after the MMTV promoter and lacks OSMR. To address the importance of OSMR pathway in the tumour microenvironment context, we injected murine cells in the mammary gland of OSMR KO and control mice.Results and discussionsThe receptor OSMR is expressed by breast cancer cells while the ligand OSM seems to be mainly produced by cancer associated macrophages and fibroblasts. Depletion of OSMR delays tumour onset, decreases tumour growth and generation of lung metastasis in MMTV-PYMT mice model. Orthotopic injections of murine TS1 cells in OSMR deficient mice shows a decrease in tumour growth compared to control mice, suggesting that OSMR signalling is also important in tumour stroma.ConclusionOur results support that OSMR pathway has an important role in the initiation and progression of breast cancer and that it is important in preparing the tumour microenvironment to facilitate tumourigenesis. OSMR could be blocked by antibody based inhibition, strategy that has had a major impact on breast cancer which makes it a promising candidate for therapeutic targeting.

Chronic expression of wild-type Ret receptor in the mammary gland induces luminal tumors that are sensitive to Ret inhibition.

The receptor tyrosine kinase Ret, a key gain-of-function mutated oncoprotein in thyroid carcinomas, has recently been implicated in other cancer types. While Ret copy number gains and mutations have been reported at low frequencies in breast tumors, we and others have reported that Ret is overexpressed in about 40% of human tumors and this correlates with poor patient prognosis. Ret activation regulates numerous intracellular pathways related to proliferation and inflammation, but it is not known whether abnormal Ret expression is sufficient to induce mammary carcinomas. Using a novel doxycycline-inducible transgenic mouse model with the MMTV promoter controlling Ret expression, we show that overexpression of wild-type Ret in the mammary epithelium produces mammary tumors, displaying a morphology that recapitulates characteristics of human luminal breast tumors. Ret-evoked tumors are estrogen receptor positive and negative for progesterone receptor. Moreover, tumors rapidly regress after doxycycline withdrawal, indicating that Ret is the driving oncoprotein. Using next-generation sequencing, we examined the levels of transcripts in these tumors, confirming a luminal signature. Ret-evoked tumors have been passaged in mice and used to test novel therapeutic approaches. Importantly, we have determined that tumors are resistant to endocrine therapy, but respond successfully to treatment with a Ret kinase inhibitor. Our data provide the first compelling evidence for an oncogenic role of non-mutated Ret in the mammary gland and are an incentive for clinical development of Ret as a cancer biomarker and therapeutic target.

Abstract 660: Pre-symptomatic detection and early treatment of mammary cancer with anti-PD-L1 in a mouse mode

Abstract Cancer kills ~8M people/year and the WHO predicts an epidemic of cancer in the developing world over the next 20 years due to increased longevity. Development of a simple method to detect and treat cancer early could be a solution to this challenge. Here we test this approach in principle in a transgenic mouse mammary tumor model by immunosignature (IMS) diagnostics. IMSs are profiles of antibodies in the blood displayed on a high density peptide array. As previous published data showed, the IMS can distinguish different diseases based on the disease specific antibody profile in the patient, including different cancers and infectious diseases. According to the immune surveillance theory, the immune system, including the humoral immune response, starts to interact with cancer cells as soon as they are transformed. First, we tested if the IMS is sensitive enough to detect the antibody profile change caused by the early tumor development in the FVB-NeuN (MMTVneu/202Mul/J) mammary cancer model. In the FVB-NeuN model, the MMTV promoter activates the wild type rat ERBB2 gene in mammary cells, and every mammary cell potentially can convert into a cancer cell. The transition from normal mammary glands to hyperplasia of FVB-NeuN in mouse starts at 8.6 to 15 weeks after birth. The mice develop the first palpable tumor in one of the ten mammary glands by 19-45 weeks of age with an average age of 32±1.2 weeks in our hands. We collected serum every 4 weeks from the FVB-NeuN mice and the wild type FVB mice for the IMS analysis. The IMS can distinguish the FVB-NeuN mouse from the age matched wild type FVB mouse as early as 12 weeks (Stage-1) before the onset of the first palpable tumor. We also analyzed the IMSs from the Stage-2 (8 weeks before the palpable tumor and so on) to Stage 4. Different IMS of each stage has only partial overlap with the other stages. The cross-stage prediction analysis showed the accuracy of the cancer detection by using different IMS is from 75% to 93%. Second, we determined if early treatment at the time of diagnosis with the checkpoint blockade, anti-PD-L1, would inhibit the tumor growth. A course of 3 injections at the time of diagnosis significantly retarded tumor growth relative to untreated controls. The early treatment elicited long-term protection which could restrict the tumor growth even four months after the treatment. The treated mice presented a specific IMS at 28 days post treatment and 35 days post the first palpable tumor, compared to the untreated mice. This indicates that the early treatment elicited an anti-tumor immune response that could inhibit tumor growth. This preliminary experiment suggests that early diagnosis combined with systemic treatment might be a method to control or eliminate cancer. Citation Format: Hu Duan, Stephen Albert Johnston, Shen Luhui. Pre-symptomatic detection and early treatment of mammary cancer with anti-PD-L1 in a mouse mode [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2017-660

Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3.

NSD3 has been implicated as a candidate driver oncogene from the 8p11-p12 locus, and we have previously published evidence for its amplification and overexpression in human breast cancer. This aim of this study was to further characterize the transforming function of NSD3 in vivo. We generated a transgenic mouse model in which NSD3 gene expression was driven by the MMTV promoter and expressed in mammary epithelium of FVB mice. Mammary glands were fixed and whole mounts were stained with carmine to visualize gland structure. Mammary tumors were formalin-fixed, and paraffin embedded (FFPE) tumors were stained with hematoxylin and eosin. Pups born to transgenic females were significantly underdeveloped compared to pups born to WT females due to a lactation defect in transgenic female mice. Whole mount analysis of the mammary glands of transgenic female mice revealed a profound defect in functional differentiation of mammary gland alveoli that resulted in the lactation defect. We followed parous and virgin NSD3 transgenic and control mice to 50weeks of age and observed that several NSD3 parous females developed mammary tumors. Whole mount analysis of the mammary glands of tumor-bearing mice revealed numerous areas of mammary hyperplasia and ductal dysplasia. Histological analysis showed that mammary tumors were high-grade ductal carcinomas, and lesions present in other mammary glands exhibited features of alveolar hyperplasia, ductal dysplasia, and carcinoma in situ. Our results are consistent with our previous studies and demonstrate that NSD3 is a transforming breast cancer oncogene.

Abstract 668: Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice

Abstract Gene amplification and mutations of cMET have been reported in a wide variety of tumors, including colorectal, lung, renal, liver, thyroid, and breast cancer. cMET(T1010I) mutation occurred in 5.2% out of 148 sibling pairs diagnosed with colorectal cancer in one study. This germline mutation occurs in about 1% of breast cancer patients, but its frequency increases to 2% in metastatic breast cancer. Furthermore, 16% of breast cancer cases with PI3KCA mutations have co-mutations of the cMET oncogene. To determine the functional consequences of this germline mutation and elucidate the molecular mechanisms of tumorigenesis induced by its oncogenic mutant protein, we developed a transgenic mouse model expressing human cMET(T1010I) under the control of a Tet-responsive promoter. MMTV promoter - directed expression of reverse tetracycline-controlled transactivator (rtTA) turns on the expression of the cMET(T1010I) transgene in murine mammary glands upon tetracycline administration. Thirty percent of these mice developed early onset mammary carcinoma within 5 months of transgene induction. This contrasts with previous reports of mammary tumor development in MMTV-cMET transgenic mice after more than a year. This strong phenotype is not the result of a random insertional effect of the transgene as no mammary tumors developed in the 29 transgene positive mice without mammary-specific expression of rtTA over a year in our study. Molecular characterization of these tumors is currently underway and the results may reveal mechanisms of tumorigenesis and novel therapeutic targets for patients harboring this mutation. In conclusion, we showed for the first time that the cMET(T1010I) mutation can trigger rapid breast tumorigenicity. By using organ-specific expression of rtTA to direct the expression of cMET(T1010I) in the organ of interest, our transgenic model may be used to understand the impact of cMET(T1010I) on many other cancer types such as colorectal, lung, renal, thyroid, and liver cancer. Citation Format: Chuanwei Yang, Ana M. Gonzalez, Naoto T. Ueno, Shuying Liu, Jinsong Zhang, Gordon B. Mills, John Mendelsohn, Prahlad Ram. Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 668.

Abstract 5095: Hic-5 modulation of the stromal matrix is required for mammary tumor progression

Abstract Primary tumors grow and invade into surrounding tissues/stroma, frequently resulting in metastasis to distant organs. Tumor cell-stroma crosstalk plays a crucial, but incompletely understood role during tumor progression and metastasis. For example, increased stromal matrix density and/or rigidity correlates with poor prognosis in human breast cancer patients. Previous studies have implicated Hic-5, a focal adhesion scaffold protein, in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in mammary tumor progression, Hic-5 -/- mice were generated and crossed with mice expressing Polyoma Middle T Antigen (PyMT) driven by the MMTV promoter. The PyMT mouse is a well-established model for human breast cancer progression. Tumors from the Hic-5 -/- PyMT mice demonstrated an increased latency and had reduced growth as compared to Hic-5 +/- PyMT controls. The absence of Hic-5 did not affect the progression of the tumor to a carcinoma stage, but there was a decrease in metastasis to the lungs. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs) and the Hic-5 -/- PyMT tumor stroma exhibits reduced extracellular matrix (ECM) deposition. Furthermore, 3D cell-derived matrices (CDM) generated in vitro using isolated CAFs, confirmed that Hic-5 -/- PyMT CAFs do not efficiently deposit and organize ECM. The organization of the stromal matrix has been shown previously to increase the rigidity of the tumor, which in turn promotes FAK Y397 phosphorylation in the tumor cells to promote cell invasion and proliferation. Accordingly, the Hic-5 -/- PyMT tumors exhibited a reduction in FAK phospho-Y397 staining and attenuation of downstream ERK activation, suggesting that their stromal matrix is not optimally organized for tumor cell signaling. Taken together, these data demonstrate that Hic-5 expression in CAFs is important for stromal matrix organization/remodeling during tumor progression and that the loss of the organization of the ECM in the Hic-5 -/- tumor stroma may contribute to the reduced tumor growth and metastasis observed in these animals. Supported by NIH CA163296 to CET, NS066071 to EO and Carol M. Baldwin Breast Cancer Fund to CET. Citation Format: Gregory J. Goreczny, Jessica Ouderkirk, Eric Olson, Mira Krendel, Christopher Turner. Hic-5 modulation of the stromal matrix is required for mammary tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5095.

Clinical applications of mouse models for breast cancer engaging HER2/neu.

Human c-ErbB2 (HER2) has long been used as a marker of breast cancer (BC) for sub-categorization for the prediction of prognosis, and determination of therapeutic strategies. HER2 overexpressing BCs are more invasive/metastatic; but patients respond to monoclonal antibody therapy with trastuzumab or tyrosine kinase inhibitors, at least at early stages. To date, numerous mouse models that faithfully reproduce HER2(+) BCs have been created in mice. We recently reviewed different mouse models of BC overexpressing wild type or mutant neu driven by MMTV, neu, or doxycycline-inducible promoters. These mice have been used to demonstrate the histopathology, oncogenic signaling pathways initiated by aberrant overexpression of HER2 in the mammary epithelium, and interaction between oncogenes and tumor suppressor genes at molecular levels. In this review, we focus on their clinical applications. They can be used to test the efficacy of HER(2) inhibitors before starting clinical trials, characterize the tumor-initiating cells that could be the cause of relapse after therapy as well as to analyze the molecular mechanisms of therapeutic resistance targeting HER2. MMTV-human ErbB2 (HER2) mouse models have recently been established since the monoclonal antibody to HER2 (trastuzumab; Herceptin®) does not recognize the rat neu protein. It has been reported that early intervention with HER2 monoclonal antibody would be beneficial for preventing mammary carcinogenesis. MDA-7/IL-24 as well as naturally-occurring chemicals have also been tested using MMTV-neu models. Recent studies have shown that MMTV-neu models are useful to develop vaccines to HER2 for immunotherapy. The mouse models employing HER2/neu will be essential for future antibody or drug screenings to overcome resistance to trastuzumab or HER(2)-specific tyrosine kinase inhibitors.

Linker histones in hormonal gene regulation

In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.

Early detection of mammary tumors in vivo using a highly specific tumor antibody.

14 Background: Earlier detection of abnormal cells using target-specific techniques holds great promise. One of those targets Mucin-1 (MUC1) is expressed as a hypo-glycosylated form (i.e., tMUC1) in 95% of breast tumors and plays a crucial role in cancer progression. We have developed Tab004, a monoclonal antibody highly specific to a protein sequence accessible in tMUC1. Here we present data assessing the specificity and sensitivity of Tab004 in vitro and in genetically engineered mice in vivo overtime. Methods: We have developed human MUC1 Tg mice that were bred to mice that carry the oncogene, polyoma middle T antigen driven by the MMTV promoter. These mice designated MMT develop mammary gland tumors spontaneously and expresses the human form of tMUC1. PyVMT and C57Bl/6 served as controls. Mice develop mammary gland hyperplasia between 8-10 weeks of age that progresses to ductal carcinoma in situ by 12-14 weeks and adenocarcinoma by 18-24 weeks. Approximately 40% of the mice develop metastasis to the lung and other organs. The tumor progression appropriately mimics the human disease. MMT mice (n = 20) were injected twice monthly retro-orbitally with 12.5ug (100uL) of Tab004-conjugated to indocyanine green (ICG) and imaged thereafter from 8 to 22 weeks of age. Fluorescence was assessed 4 and 24 hrs post injection using the IVIS system. At euthanasia, tissue was collected for further analyses. Further, human breast tumor and normal mammary epithelial tissues were evaluated by immunohistochemical staining. Results: Tab004 specifically recognizes tMUC1 and not normal MUC1. In MMT mice, ICG-conjugated Tab004 allowed early detection of tumors in vivo sparing recognition of normal mammary epithelia in the C57BL/6 mice or in the PyV MT tumors. Detection with ICG-conjugated Tab004 allowed monitoring of tumor progression overtime. Importantly, ICG-conjugated Tab004 permitted significantly earlier detection than physical examination. Conclusions: The data highlight the specificity and the sensitivity of Tab004 in detecting tMUC1 in vitro, in situ and in relevant murine models in vivo. Thus, Tab004 will have significant clinical relevance for development as a targeted imaging agent and in the future for targeted drug delivery.

Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in Vitro Reveal Exchange on the Second Time Scale.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that interactions of GR with chromatin are highly dynamic, with average receptor residence times of only seconds. These findings were surprising because early kinetic studies found that GR-DNA interactions in vitro were much slower, having calculated residence times of minutes to hours. However, these latter analyses were conducted at a time when it was possible to work with only either partially purified holoreceptor or its purified but isolated DNA binding domain. Noting these limitations, we reexamined GR-DNA dissociation kinetics using a highly purified holoreceptor shown to be amenable to rigorous study. We first observe that GR-DNA interactions in vitro are not slow as previously thought but converge with in vivo behavior, having residence times of only seconds to tens of seconds. This rapid exchange is seen at six individual response elements and the multisite MMTV promoter used in live cell imaging. Second, GR dissociation rates are identical for all response elements. Thus, previously observed differences in receptor affinity toward these sequences are not due to differences in off rate but in on rate. Finally, dissociation kinetics are biphasic in character. A minimal kinetic model consistent with the data is that in which DNA-bound GR interconverts between states on a second time scale, with dissociation occurring via a multistep process. We speculate that receptor interconversion in this time frame can be recognized by the coregulatory proteins that interact with GR, leading to unique transcriptional responses.

Abstract 3461: RSK2 is a driver of estrogen receptor positive breast cancer

Abstract For unknown reasons, high levels of active Ser/Thr protein kinase RSK in estrogen receptor α (ERα)-positive (ER+) breast cancer patients correlate with better responsiveness to anti-estrogen therapies. Investigation of these surprising observations led us to discover an unanticipated mechanism in which ERα controls RSK2 nuclear accumulation. RSK2 is retained in the nucleus by physical association with ERα and this interaction is disrupted by anti-estrogen treatment. Consistent with these findings, in primary human ER+ breast tissue organoids, 4-hydroxy-tamoxifen (4-OHT) drove export of RSK2 to the cytoplasm. Importantly, RSK2 nuclear accumulation is critical for the expression of the oncogene, cyclin D1. This conclusion is supported by the observations that a RSK2 mutant, which cannot accumulate in the nucleus, does not rescue cyclin D1 in cells silenced for endogenous RSK2. Analysis of RNAseq data shows that ERα and RSK2 cooperatively regulate a transcriptional program, which includes cyclin D1. The physiological relevance of these conclusions is supported by our observations that in ∼60% of ER+ breast cancer patients, active nuclear RSK correlates with ERα and cyclin D1 levels. Taken together, these findings suggest that responsiveness to endocrine-based therapy is driven by reduced RSK2 nuclear accumulation, which in turn reduces expression of cyclin D1 and of other tumor-promoting effectors. Based on these observations we hypothesized that nuclear RSK2 cooperates with ERα and is a physiologically relevant driver of ER+ breast cancer. To test this hypothesis we generated a transgenic mouse model in which the MMTV promoter controls expression of RSK2 fused with the nuclear localization signal (NLS) of SV40. Strikingly, in ∼50% of transgenic mice forced nuclear accumulation of RSK2 in the mammary gland caused hyperplasia and ductal carcinoma in situ (DCIS). These results are the first demonstration that RSK2 can drive proliferation and transformation in vivo. Transgenic animals had a 4-fold increase in the number of cyclin D1 expressing cells and a 6-fold increase in Ki67 levels. Surprisingly, in vivo expression of nuclear RSK2 expanded the population of ER+ cells. These results demonstrate, for the first time, that nuclear RSK2 drives the amplification of the cell population relevant to ER+ breast cancer. In agreement with these observations we found that overexpression of ERα in the mouse mammary gland expands exclusively, by ∼3 fold, the cell population that co-expresses ERα and active nuclear RSK. These results further support our hypothesis that RSK2 cooperates with ERα and is an important drug target for ER+ breast cancer. Citation Format: Katarzyna Ludwik, Deborah Lannigan. RSK2 is a driver of estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3461. doi:10.1158/1538-7445.AM2015-3461