Mouse Mammary Tumor Model Research Articles

Development and Preclinical Application of an Immunocompetent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is associated with a poor prognosis and for which no targeted therapies currently exist. In order to improve preclinical testing for TNBC that relies primarily on using human xenografts in immunodeficient mice, we have developed a novel immunocompetent syngeneic murine tumor transplant model for basal-like triple-negative breast cancer. The C3(1)/SV40-T/t-antigen (C3(1)/Tag) mouse mammary tumor model in the FVB/N background shares important similarities with human basal-like TNBC. However, these tumors or derived cell lines are rejected when transplanted into wt FVB/N mice, likely due to the expression of SV40 T-antigen. We have developed a sub-line of mice (designated REAR mice) that carry only one copy of the C3(1)/Tag-antigen transgene resulting from a spontaneous transgene rearrangement in the original founder line. Unlike the original C3(1)/Tag mice, REAR mice do not develop mammary tumors or other phenotypes observed in the original C3(1)/Tag transgenic mice. REAR mice are more immunologically tolerant to SV40 T-antigen driven tumors and cell lines in an FVB/N background (including prostate tumors from TRAMP mice), but are otherwise immunologically intact. This transplant model system offers the ability to synchronously implant the C3(1)/Tag tumor-derived M6 cell line or individual C3(1)/Tag tumors from various stages of tumor development into the mammary fat pads or tail veins of REAR mice. C3(1)/Tag tumors or M6 cells implanted into the mammary fat pads spontaneously metastasize at a high frequency to the lung and liver. M6 cells injected by tail vein can form brain metastases. We demonstrate that irradiated M6 tumor cells or the same cells expressing GM-CSF can act as a vaccine to retard tumor growth of implanted tumor cells in the REAR model. Preclinical studies performed in animals with an intact immune system should more authentically replicate treatment responses in human patients.

Combined administration of the apoptin gene and poly (I:C) induces potent anti-tumor immune response and inhibits growth of mouse mammary tumors

BackgroundMany viral proteins exhibit selective cytotoxicity for tumor cells without affecting the normal diploid cells. The apoptin protein of chicken infectious anemia virus is one of such proteins, which has been shown to kill tumor cells specifically. However, an effective cancer treatment strategy also requires assistance from the immune system. Recently, poly (I:C) has been shown to be an effective cancer vaccine adjuvant. AimIn this study, we assessed the anti-tumor potential of apoptin gene transfer alone and in combination with poly (I:C) in a 4T1 mouse mammary tumor model. Methods4T1 cells were used to induce mammary tumor in Balb/c mice. Mice bearing tumors were divided into 6 groups, and each group received six intratumoral injections during a period of one month. After the last immunization, the animals were sacrificed, and peripheral blood, spleen, lungs, liver, heart, kidney and tumor tissues were collected for immunological, molecular and pathological analysis. ResultsWe report that intratumoral administration of apoptin plasmid along with poly (I:C) not only significantly inhibited the growth of mammary tumor, but also induced a potent anti-tumor immune response as indicated by the increase in blood CD4+, CD8+ cells and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed increased secretion of Th1 cytokines (IFN-γ and IL-2). ConclusionsThe results of our study demonstrate that the inclusion of poly (I:C) significantly enhanced the anti-tumor activity of apoptin mainly by inducing a potent anti-tumor immune response. Therefore, we report the use of apoptin and poly (I:C) combination as a novel and powerful strategy for cancer immunotherapy.

Abstract B05: Repurposing FDA-approved drugs for anti-metastatic therapy: Results of an integrated genome wide screen for metastasis modifiers

Abstract Metastasis remains the primary cause of patient morbidity and mortality in solid tumors. Recent advances in genomic technologies have provided major insights into the etiology of tumors. However, significantly less is known about factors that contribute to metastasis. Here we report the results of a genome-wide integrated genetic, transcriptional, and epigenetic analysis in a mouse mammary tumor model to identify factors associated with tumor progression. This strategy identified a prognostic transcription signature and several upstream transcriptional regulators for metastatic disease. Interestingly, this analysis also identified novel or other FDA-approved drugs as potentially useful for anti-metastatic therapy. As predicted by this screen, administration of antidiabetic thiazolidones Rosiglitazone and Pioglitazone suppressed metastasis in an orthotopic breast cancer mouse model. Similarly, ciprofloxacin, an antibiotic, also decreased breast cancer metastasis. This strategy is thus not only of prognostic value but also presents the opportunity to rapidly repurpose drugs with proven safety and efficacy profiles for control and treatment of advanced neoplastic disease. Citation Format: Anjali Shukla, Kent W. Hunter. Repurposing FDA-approved drugs for anti-metastatic therapy: Results of an integrated genome wide screen for metastasis modifiers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B05.

Abstract IA20: Genomics of a STAT1 knockout mouse model of human ER+ breast cancer

Abstract Estrogen receptor alpha positive luminal breast cancers are the most frequent subtype of breast cancer. Previous work has established that Stat1-/- mouse mammary tumor model recapitulates signaling, expression and phenotypic alterations observed in this subtype of human breast cancers. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of 22 Stat1-/- primary mammary tumors and cell lines. We discovered a novel hotspot of somatic mutations in 100% of tumors that resulted in a truncated form of the prolactin receptor (Prlr). Targeted sequence analysis identified similar mutations in 77.8% of ductal carcinoma in situ. Co-expression of truncated and full-length Prlr in normal cells led to activation of oncogenic Stat3 and Stat5 as well as cellular transformation. In conclusion, truncating mutations of Prlr drive tumor development in a model of human ERa+ breast cancer and should be considered as novel antitumor targets. Citation Format: Elaine Mardis, Obi L. Griffith, Ruby Chan Szeman, Malachi Griffith, Kilannin Krysiak, Zachary Skidmore, Jasreet Hundal, Julie A. Allen, Arthur Cora, Alexander P. Miceli, Heather Schmidt, Lee Trani, Krishna-Latha Kanchi, Christopher A. Miller, David E. Larson, Robert S. Fulton, Richard K. Wilson, Robert D. Schreiber. Genomics of a STAT1 knockout mouse model of human ER+ breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA20.

Abstract A05: The tumor suppressor function of Plk2 in triple-negative breast cancer may be mediated through regulation of Plk1

Abstract Triple-negative breast cancers (TNBCs) are highly aggressive, associated with poor prognosis and lack targeted therapies. Current breast cancer therapies target the estrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors, which are absent in TNBCs. Developing new treatment strategies for TNBCs requires a better understanding of the signaling networks regulating TNBCs. Polo-like kinase 1 (Plk1) is a putative oncogene in TNBC. Plk1 is frequently overexpressed and promotes mitotic cell division, making it an attractive target for cancer therapy. Several inhibitors of Plk1 exist, one of which has accelerated to phase III clinical trials for acute myeloid leukemia. However, these drugs also inhibit Plk2, another polo-like kinase family member. The impact that the presence of Plk2 has on the effectiveness of Plk1 inhibitors as a cancer therapy is unknown. We reported recently that a loss of Plk2 in the developing mammary gland results in increased proliferation, hyperbranching, misoriented mitotic spindle assembly and defects in polarity (Villegas et al Development 2015). Loss of Plk2 was accompanied by increased expression of Plk1. Genetic rescue experiments, knocking down Plk1 in Plk2 null mouse mammary epithelium, and bimolecular fluorescence complementation assays, using wildtype Plk2 and a kinase dead mutant (KD) of Plk2 as bait, revealed that Plk2 regulates these processes through its direct interaction with Plk1. Our preliminary data suggest that loss of Plk2 results in increased Plk1 protein but not RNA expression. We propose that Plk2 functions as a tumor suppressor by decreasing Plk1 stability in TNBCs. Loss of Plk2, therefore, may sensitize tumors to treatment with Plk1 inhibitors if these tumors display Plk1 oncogene addiction. We hypothesize that Plk2, through targeted degradation of Plk1, inhibits tumorigenesis in TNBC. We observed that loss of Plk2 alone is not sufficient to generate mouse mammary tumors, however more lesions form after multiple pregnancies in Plk2 null glands than wildtype. To examine the tumor suppressor function of Plk2 through its regulation of Plk1 in TNBC, we are generating preclinical mouse mammary tumor models integrating the germline loss of Plk2 with p53 loss or c-Myc overexpression frequently observed in TNBC. Finally, to investigate the clinical relevance of Plk2 in TNBC, we will use available tissue microarrays of TNBC patient derived xenograft (PDX) mouse models to identify those that exhibit loss of Plk2. We will treat the identified PDX models with Plk1 inhibitors to confirm that Plk2 loss promotes Plk1 in human TNBC samples. With these studies, we expect to find that Plk2 is involved in the targeted degradation of Plk1 in TNBC, sensitizing this aggressive breast cancer subtype to treatment with Plk1 inhibitors. The results of these studies should help validate whether Plk2 is a new biomarker for determining which patients will benefit from Plk1 targeted TNBC treatment. Supported by Susan G. Komen Foundation grant SAC110031. Citation Format: Deanna Acosta, Elizabeth Villegas, Elena Kabotyanski, Celina Montemayor, Sarah J. Kurley, Rocio Dominguez-Vidana, Chad A. Shaw, Thomas F. Westbrook, Jeffrey M. Rosen. The tumor suppressor function of Plk2 in triple-negative breast cancer may be mediated through regulation of Plk1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A05.

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

TLR4/IFNγ pathways induce tumor regression via NOS II-dependent NO and ROS production in murine breast cancer models

ABSTRACTToll-like receptor (TLR) 4 agonists have emerged as a new group of molecules used for cancer therapy. They have been exploited to enhance the immunogenicity of current chemotherapeutic regimens. However, their effects on cancer cells remain elusive. Here, we showed that a TLR4 agonist, namely a synthetic lipid A analog (ALA), OM-174, exhibits antitumor effects in several mammary tumor mouse models. We also showed that immune components are involved in such effects, as attested to by the failure of ALA to induce tumor regression or an increase of animal survival in mice knocked-out for interferon γ (IFNγ) or TLR4. TLR4 and IFNγ receptor (INFR2) expressed by cancer cells are involved in the antitumor efficacy of ALA since this last did not inhibit tumor growth in mice bearing a tumor but lacking TLR4 or IFNγ receptor 2 (IFNR2). Mechanistic investigations revealed that nitric oxide (NO), superoxide and peroxynitrite produced by uncoupling of inducible NO synthase (NOS II) in cancer cells are key mediators of ALA and IFNγ-mediated tumor growth inhibition. We present here a comprehensive picture of tumor cell death induction, in vivo and in vitro, by immunotherapy and for the first time the involvement of the TLR4/IFNγ/NOS II pathway in immunotherapy was investigated.

Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer.

IntroductionDespite advances in early detection and adjuvant targeted therapies, breast cancer is still the second most common cause of cancer mortality among women. Tumor recurrence is one of the major contributors to breast cancer mortality. However, the mechanisms underlying this process are not completely understood. In this study, we investigated the mechanisms of tumor dormancy and recurrence in a preclinical mouse model of breast cancer.MethodsTo elucidate the mechanisms driving tumor recurrence, we employed a transplantable Wnt1/inducible fibroblast growth factor receptor (FGFR) 1 mouse mammary tumor model and utilized an FGFR specific inhibitor, BGJ398, to study the recurrence after treatment. Histological staining was performed to analyze the residual tumor cells and tumor stroma. Reverse phase protein array was performed to compare primary and recurrent tumors to investigate the molecular mechanisms leading to tumor recurrence.ResultsTreatment with BGJ398 resulted in rapid tumor regression, leaving a nonpalpable mass of dormant tumor cells organized into a luminal and basal epithelial layer similar to the normal mammary gland, but surrounded by dense stroma with markedly reduced levels of myeloid-derived tumor suppressor cells (MDSCs) and decreased tumor vasculature. Following cessation of treatment the tumors recurred over a period of 1 to 4 months. The recurrent tumors displayed dense stroma with increased collagen, tenascin-C expression, and MDSC infiltration. Activation of the epidermal growth factor receptor (EGFR) pathway was observed in recurrent tumors, and inhibition of EGFR with lapatinib in combination with BGJ398 resulted in a significant delay in tumor recurrence accompanied by reduced stroma, yet there was no difference observed in initial tumor regression between the groups treated with BGJ398 alone or in combination with lapatinib.ConclusionThese studies have revealed a correlation between tumor recurrence and changes of stromal microenvironment accompanied by altered EGFR signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0649-1) contains supplementary material, which is available to authorized users.

Trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules through engagement of natural killer cells and stimulation of IFNγ secretion

ABSTRACTIt is increasingly recognized that trastuzumab, an antibody approved for treating human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, exerts some of its antitumor effects through enhanced T cell responses. Full activation of CD8+ T cells requires both expression of major histocompatibility complex class I molecules (HLA-ABC) and expression of the T cell costimulatory molecule CD80 or CD86; however, the impact of trastuzumab treatment on the expression of HLA-ABC and CD80 and CD86 has not been investigated in HER2-overexpressing breast cancer cells. In this study, we found that, while there is no direct correlation between the expression of HER2 and HLA-ABC in breast cancer, knockdown of HER2 or inhibition of HER2 kinase by lapatinib downregulated HLA-ABC expression. Trastuzumab had no meaningful effects on HLA-ABC expression in HER2-overexpressing breast cancer cells in monoculture; however, treatment of such cells with trastuzumab in co-culture with human peripheral blood mononuclear cells (PBMC) significantly upregulated not only HLA-ABC expression but also CD86 expression. We showed that this upregulation was mediated by interferon gamma (IFNγ) secreted from the natural killer (NK) cells in PBMC as a result of engagement of NK cells by trastuzumab. We further confirmed this effect of trastuzumab in vivo using a mouse mammary tumor model transduced to overexpress human HER2. Together, our data provide evidence that trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules in HER2-overexpressing breast cancer cells in the presence of PBMC, which supports the view that T-cell-mediated immune responses are involved in trastuzumab-mediated antitumor effects.

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual's predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency.

Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan’s National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

Abstract 1352: Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development

Abstract Preventative vaccine therapy may benefit women at high risk for breast cancer but currently it is impossible to identify women who will develop breast cancer prior to tumor development to identify initiating cancer antigens. Immunocompetent transgenic mouse mammary tumor models that are genetically similar to human breast cancer subtypes may identify pre-invasive antigens because mice can be followed longitudinally. Comparing tumor growth characteristics in two mouse models (TgMMTV-neu is genetically similar to luminal B breast cancer and C3(1)tag is genetically similar to triple negative breast cancer) demonstrated that, similar to triple negative breast cancer, C3(1)Tag mice develop tumors earlier and tumors grow more rapidly than TgMMTV-neu tumors. Furthermore, C3(1)Tag tumors have increased CD8+/CD4+ ratio (p<0.05) similar to human triple negative tumors. Using these two mouse models, we identified putative pre-invasive breast cancer antigens present prior to tumor development by serologic analysis of recombinant cDNA expression libraries and serological analysis of chip-arrayed proteins. We identified 65 pre-invasive antigens that were present in mice that would develop cancer but not parental control mice. The goal of this study was to identify which of the 65 antigens were required for human breast cancer survival with the goal to develop a preventative multi-antigen polyepitope breast cancer vaccine. We chose the pre-invasive antigens necessary for human breast cancer tumor cell survival using a high throughput siRNA screen evaluating for increased apoptosis and decreased cell survival in either HER2 positive or triple negative human breast cancer cell lines with decreased expression of the target protein. Five of the antigens were essential for human breast cancer cell survival: VPS35, SERBP1, ARPC2, PDIA6, and KRT8. All of these genes have roles in human cancer progression, and KRT8, SERBP1, and PDIA6 have identified roles in breast cancer pathogenesis. After designing human MHC class II peptides for each of these targets that cover at least 25% of the protein, we evaluated implanted tumor inhibition in the mouse models. Vaccination VPS35 peptides inhibited tumor growth by 47% (p<0.0001), vaccination with ARPC2 peptides inhibited tumor growth by 54% (p<0.0001), and vaccination with SERBP1 peptides inhibited tumor growth by 61% (p<0.0001) in the TgMMTV-neu mice. In C3(1)Tag mice, vaccination with VPS35 peptides inhibited tumor growth by 39% (p<0.0001) and vaccination with SERBP1 peptides inhibited tumor growth by 59% (p<0.0001) but vaccination with ARPC2 peptides did not inhibit tumor growth. These studies have demonstrated that mouse models can be used to identify pre-invasive breast cancer antigens. Further studies will evaluate the use of vaccines containing epitopes from several of the antigens to prevent spontaneous tumors in these mouse models. Citation Format: Sasha E. Stanton, Ekram Gadd, Lauren Rastetter, James Annis, Jianning Mao, John Ladd, Samir Hanash, Mary L. Disis. Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development. [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 1352. doi:10.1158/1538-7445.AM2015-1352

Abstract 279: Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu

Abstract In breast cancer, increased immune infiltrate prior to neoadjuvant chemotherapy predicts improved pathologic complete response (pCR) and improved survival, however almost half of breast tumors have no CD8+ T cell infiltrate. Evidence has emerged that conventional breast cancer chemotherapy can increase CD8+ T cells (doxorubicin) and decrease the immunosuppressive regulatory CD4+ T cells (paclitaxel and cyclophosphamide); therefore discovering well tolerated agents that further enhance the anti-tumor immune function of these chemotherapies may further improve response in breast cancer patients. The oral agent bexarotene (a retinoic receptor agonist) showed a 20% disease response as a single agent in metastatic breast cancer and has been shown to increase CD8+ T cell tumor infiltration and decrease CD8+ T cell apoptosis in vitro. The goal of this study is to demonstrate whether this well tolerated oral agent can enhance the anti-tumor response of conventional chemotherapy through increasing intratumoral CD8+ T cells in TgMMTV-neu transgenic mice that are immunologically competent and genetically similar to human luminal B breast cancer. Two of the most active chemotherapies in human breast cancer, doxorubicin and paclitaxel, each could inhibit tumor growth by 70-80% and increase CD8+ T cell tumor infiltrate by approximately 40% in tumor bearing TgMMTV-neu mice, a third breast cancer chemotherapy cyclophosphamide increased CD8+ tumor infiltrate by 45% (p = 0.011) but did not show a statistically significant decrease in tumor volume. When TgMMTV-neu mice with ∼100 mm3 tumors were treated with 50 mg/m2 bexarotene for four weeks they demonstrated a 44% increase in CD8+ tumor infiltrate (p = 0.05) and 60% decrease in mean tumor growth (p = 0.03) compared to control. However, the addition of bexarotene to chemotherapy was superior to chemotherapy alone, and significantly more effective than bexarotene alone. This enhanced anti-tumor function was even seen with cyclophosphamide that by itself had not inhibited tumor growth. Adding bexarotene to weekly doxorubicin decreased tumor growth by 98% (p = 0.008 compared to doxorubicin alone), adding bexarotene to weekly paclitaxel decreased tumor growth by 86% (p = 0.02 compared to paclitaxel alone), and adding bexarotene to weekly cyclophosphamide inhibited tumor growth by 82% (p = 0.04 compared to cyclophosphamide alone). Further studies are now ongoing to identify the anti-tumor role of bexarotene particularly its immune modulatory role. These results suggest that the addition of bexarotene, a relatively well-tolerated oral agent, may modify the immune environment and improve tumor response to chemotherapy in breast cancer possibly improving response to neoadjuvant chemotherapy. Citation Format: Sasha E. Stanton, Ekram Gadd, Edmond Marzbani, Lauren Rastetter, Mary L. Disis. Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu. [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 279. doi:10.1158/1538-7445.AM2015-279

Abstract SY38-03: Targeting post-translational activation of MYC for the treatment of cancer

Abstract The MYC oncoprotein is considered to be one of the most important targets in cancer; however, successful targeting of MYC has not been achieved to date. MYC is a transcription factor, which binds to DNA in target genes as a heterodimer with its partner protein, MAX. While MAX is constitutively expressed, MYC protein expression is tightly regulated at multiple levels, including gene transcription, mRNA stability, protein translation, and post-translational protein stability. In cancer, MYC expression is upregulated through deregulation at many of these levels, resulting in an increase in MYC:MAX heterodimers and MYC-regulated gene expression. MYC regulated genes are involved in nearly all cellular processes, including cell intrinsic properties such as cell cycle control, cell growth and metabolism, self-renewal, migration and invasion, as well as cell extrinsic programs including angiogenesis, stromal cell expansion, and alterations in immune cell surveillance. MYC is thus a master regulator of cellular phenotype, and its deregulation contributes to tumorigenesis through many mechanisms. Importantly, elegant studies have recently demonstrated the tractability of targeting MYC for cancer therapeutics. Specifically, loss of MYC in adult tissues caused very little toxicity and metronomic expression of a MYC dominant negative protein caused no observable toxicity, but eliminated KRAS-driven pancreatic and lung tumors. Since targeting MYC directly has proven difficult, we are developing new strategies to target the post-translational activation of MYC. MYC is both stabilized and transcriptionally activated following cell stimulation via receptor tyrosine kinase signaling pathways leading to ERK mediated phosphorylation of Serine 62. We have demonstrated that Serine 62 phosphorylated MYC is upregulated in human cancer cells and that this facilitates its regulation of pro-oncogenic target genes by increasing its DNA binding activity and co-activator recruitment, as well as increasing its protein stability. We have identified Protein Phosphatase 2A (PP2A) as the enzyme responsible for dephosphorylating Serine 62. PP2A is a critical tumor suppressor that is known to inactivate multiple oncogenic signaling pathways, as well as cell cycle drivers and survival factors. Furthermore, inactivation of PP2A has been shown to be critical for the transformation of human cells, and PP2A activity is suppressed in most tested human tumors. We are utilizing a recently developed small molecule, orally available, allosteric PP2A activator drug. This drug (DTx) is in clinical development by Dual Therapeutics. We have tested this drug, as well as a peptide mimetic PP2A activator, OP449, in cell lines and in mouse models of tumorigenesis. We have observed cytotoxic activity in both breast and pancreas cancer cell lines associated with dephosphorylation of MYC at Serine 62. We have also observed reduced MYC DNA target gene binding and target gene expression with PP2A activation therapy. In order to test novel therapeutic strategies targeting MYC activity, we have developed new mouse models of Myc-driven tumorigenesis using our ROSA26-LSL-Myc mice that we generated to conditionally express transcriptionally deregulated, physiological levels of Myc in response to Cre recombinase. By crossing these mice with mice expressing other organ specific oncogenic drivers, we have developed mouse mammary tumor models representing HER2+ and Triple Negative breast cancer. We have also developed a novel mouse model of pancreatic tumorigenesis using the same strategy. All of these models appear to molecularly recapitulate the corresponding human disease, and we are using them as pre-clinical testing platforms for our PP2A activation therapy trials. So far, we have seen dramatic tumor growth inhibition and even tumor shrinkage with DTx treatment in all three of these models. This is associated with loss of Serine 62 phosphorylation in the treated tumors. Importantly, we see no signs of toxicity in vivo with this novel compound and pharmacokinetic studies look very promising for bringing this compound to the clinic. In summary, we believe that targeting pathways that post-translationally activate MYC's oncogenic potential is a promising strategy for targeting this important oncoprotein. Citation Format: Xiaoyan Wang, Amy Farrell, Mahnaz Janghorban, Brittany Allen-Petersen, Juan Liang, Tyler Risom, Michael Ohlmeyer, Goutham Narla, Rosalie C. Sears. Targeting post-translational activation of MYC for the treatment of 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 SY38-03. doi:10.1158/1538-7445.AM2015-SY38-03

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress-mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress-mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc-overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC-overexpressing breast cancers.

Adoptive therapy of ROR1+ murine solid tumors with chimeric antigen receptor-modified T cells (VAC7P.1045)

Abstract Adoptive therapy with T cells modified to express chimeric antigen receptors (CARs) holds promise for treating epithelial cancers if target molecules can be identified and strategies developed to overcome the immunosuppressive mechanisms of solid tumors. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed in many blood and solid tumors but is absent from vital adult tissues, making it a potential tumor-specific target for therapy. We developed ROR1-specific CARs comprised of an extracellular single chain antibody linked to various intracellular signaling modules. T cells modified to express ROR1-CARs exerted potent anti-tumor effects against ROR1+ leukemia and lymphoma in vivo, but their effectiveness against solid tumors is not known. To test this, we used the orthotopic 4T1 mouse mammary tumor model that recapitulates the immunosuppressive microenvironment and metastatic pattern of human breast cancer. We transduced 4T1 cells with firefly luciferase (ffluc) to allow in vivo imaging of tumor growth, along with mouse or human ROR1. Murine CD8+ T cells modified to express ROR1-CARs lysed ROR1+ but not ROR1- 4T1 cells in vitro, while unmodified T cells were unable to lyse either ROR1+ or ROR1- 4T1 cells. We are currently evaluating the anti-tumor activity of ROR1-CAR T cells against 4T1-ffluc/ROR1 cells in vivo to define optimal CAR constructs and cell compositions and identify what immunosuppressive pathways to target to improve anti-tumor activity.

Abstract P1-07-28: Expression of S100A14 promotes cancer cell invasion and metastasis and is associated with poor prognosis in breast cancer

Abstract Introduction: Identifying key molecules involved in cancer metastasis is important in anticancer drug discovery. To identify such molecules, we used a dual approach involving: 1) gene screening and in vivo confirmation of metastasis-promoting molecules using a mouse mammary tumor model; and 2) functional and clinicopathological analyses of the candidate molecules in a human cancer. The products of several candidate genes promoted metastasis of mouse mammary tumors. A protein designated S100A14 promoted metastasis in the mouse model and was found to be associated with poor prognosis in human breast cancer patients. Methods and Results: 1) Identification of metastasis-related molecules: We established two highly metastatic cell lines, 66Lu10 and 66HM, which formed multiple metastatic colonies in general organs, from MCH66 mouse mammary tumor cells. Two low-metastasis cell lines, 66C8 and 66LM1, were also established. DNA microarray analysis was used to identify candidate metastasis-related genes based on differential expression in 66Lu10 and 66HM cells compared with 66C8 and 66LM1. An in vivo metastasis assay in which the highly metastatic cells were transfected with gene-specific siRNAs and orthotopically inoculated into mice was performed to confirm the metastasis-promoting activity of the candidate genes. Several novel molecules that promoted metastasis in the mouse model were identified, including S100A14. 2) Significance of S100A14 in human cancer: Analysis of S100A14 protein expression in various human cancer cell lines and tissues revealed that S100A14 expression is upregulated in breast cancer. Functional analysis using MCF7 and SK-BR-3 human breast cancer cells showed that S100A14 colocalizes with F-actin on the cell membrane at cell-cell attachment sites and on focal adhesions at the leading edge. Immunoprecipitation analysis also demonstrated an interaction between S100A14 and F-actin. Boyden chamber and wound-healing assays showed that S100A14 knockdown substantially suppresses the invasive activity of both cell lines. Immunohistochemical analysis of archival specimens of primary tumors from 167 breast cancer patients showed strong membranous staining of S100A14 (53%). Elevated expression of this protein was significantly associated with younger age (<60 years), ER-negative status, HER2-positive status, and poorer prognosis (P=0.0002). Conclusions: Using our mouse mammary tumor model for identifying metastasis-related genes, we identified S100A14 as a strong candidate metastasis-promoting molecule in vivo. Functional analyses using human breast cancer cell lines indicated that S100A14 promotes invasive activity via interaction with cytoskeletal components. Upregulated expression of S100A14 is associated with poor prognosis in breast cancer patients. Therefore, S100A14 is a potential prognostic biomarker and breast cancer therapeutic target. Citation Format: Takashi Sugino, Mizuko Tanaka, Takuma Oishi, Takashi Nakajima. Expression of S100A14 promotes cancer cell invasion and metastasis and is associated with poor prognosis in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-28.