Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Currently, an in vivo model of human breast cancer metastasizing from the orthotopic site to bone does not exist, making it difficult to study the many steps of skeletal metastasis. Moreover, models used to identify the mechanisms by which breast cancer metastasizes to bone are limited to intracardiac injection, which seeds the cancer cells directly into the circulation, thus bypassing the early steps in the metastatic process. Such models do not reflect the full process of metastasis occurring in patients. We have developed an animal model of breast cancer metastasis in which the breast cancer cells and the bone target of osteotropic metastasis are both of human origin. The engrafted human bone is functional, based on finding human IgG in the mouse bloodstream, human B cells in the mouse spleen, and normal bone histology. Furthermore, orthotopic injection of a specific human breast cancer cell line, SUM1315 (derived from a metastatic nodule in a patient), later resulted in both bone and lung metastases. In the case of bone, metastasis was to the human implant and not the mouse skeleton, indicating a species-specific osteotropism. This model replicates the events observed in patients with breast cancer skeletal metastases and serves as a useful and relevant model for studying the disease.

BackgroundMetastasis in breast cancer foreshadows mortality, as clinically evident disease is aggressive and generally chemoresistant. Disseminated breast cancer cells often enter a period of dormancy for years to decades before they emerge as detectable cancers. Harboring of these dormant cells is not individually predictable, and available information suggests that these micrometastatic foci cannot be effectively targeted by existing therapies. As such, long-term, relatively non-toxic interventions that prevent metastatic outgrowth would be an advance in treatment. Epidemiological studies have found that statins reduce breast cancer specific mortality but not the incidence of primary cancer. However, the means by which statins reduce mortality without affecting primary tumor development remains unclear.MethodsWe examine statin efficacy against two breast cancer cell lines in models of breast cancer metastasis: a 2D in vitro co-culture model of breast cancer cell interaction with the liver, a 3D ex vivo microphysiological system model of breast cancer metastasis, and two independent mouse models of spontaneous breast cancer metastasis to the lung and liver, respectively.ResultsWe demonstrate that statins can directly affect the proliferation of breast cancer cells, specifically at the metastatic site. In a 2D co-culture model of breast cancer cell interaction with the liver, we demonstrate that atorvastatin can directly suppress proliferation of mesenchymal but not epithelial breast cancer cells. Further, in an ex vivo 3D liver microphysiological system of breast cancer metastasis, we found that atorvastatin can block stimulated emergence of dormant breast cancer cells. In two independent models of spontaneous breast cancer metastasis to the liver and to the lung, we find that statins significantly reduce proliferation of the metastatic but not primary tumor cells.ConclusionsAs statins can block metastatic tumor outgrowth, they should be considered for use as long-term adjuvant drugs to delay clinical emergence and decrease mortality in breast cancer patients.

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Metastasis is the ultimate cause of mortality in breast cancer patients. Critical to our ability to prevent and treat metastasis is the identification of the key factors mediating the metastatic process and the understanding of their biological function. Using a murine model of breast cancer metastasis, we recently identified the transcriptional co-activator CITED2 as a potential mediator of bone metastasis. Here, we further explore the potential role of CITED2 in facilitating metastasis using human breast cancer cells. First, we examined the expression of CITED2 mRNA in human breast cancer patient samples by qRT-PCR. Interestingly, CITED2 mRNA levels were significantly (p < 0.05) elevated in primary breast tumors from patients surviving less than 5 years from time of diagnosis (non-survivors, n = 8) relative to those who survived greater than 5 years (survivors, n = 11). Further, mRNA levels of CITED2 were significantly (p < 0.05) elevated in breast cancer metastases (n = 25) relative to both primary breast tumors from survivors and normal mammary organoids. Consistent with mRNA expression, CITED2 protein levels were elevated in primary breast tumors and metastases relative to normal mammary epithelium by immunohistochemical analysis. To investigate the function of CITED2 we generated cell lines in which CITED2 expression was stably increased (CITED2) or decreased (shCITED2) using the human breast cancer cell line MDA-MB-231. While increased expression of CITED2 did not affect cell proliferation in vitro by MTS assay, survival following intracardiac injection was significantly reduced (p < 0.01) suggesting that CITED2 may enhance the ability of cancer cells to survive and grow at secondary sites. Consistent with this hypothesis, we also observed enhanced osteolysis at sites of bone metastasis in animals harboring CITED2 cells relative to vector control. In contrast, stable reduction of CITED2 levels significantly (p < 0.05) inhibited cell proliferation in vitro by MTS assay, consistent with the transcriptional regulation of numerous genes controlling cell cycle events (eg. cyclin E, cdk1, p21, ATM) as observed by qRT-PCR. Moreover, reduced expression of CITED2 resulted in the reversion of MDA-MB-231 cells to a more epithelial-like phenotype as evidenced by a more cuboidal morphology under light microscopy. This observation was further supported by Western analysis demonstrating decreased protein expression of the mesenchymal marker vimentin and increased expression of the epithelial markers E-cadherin and claudin-3. Further, reduction of CITED2 expression inhibited invasive ability by Matrigel invasion assay, while increased levels of CITED2 enhanced invasion. Taken together, these data support a potential role for CITED2 in regulating the metastatic potential of breast cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3394.

Breast cancer is the most diagnosed cancer type in women worldwide. It is a heterogeneous disease that can progress and metastasize into distant organs. After lung cancer, breast cancer is the second leading cause of cancer death among women in developed countries. However, death rates are declining since 1990, mainly due to earlier detection and improved therapies. The development of therapies specifically targeting known oncogenes have improved patient survival. For example the estrogen receptor (ER), a hormone receptor expressed in 70% of breast cancer and the receptor tyrosine kinase ErbB2, which is expressed in 20% of breast cancer, are treated by endocrine therapies and targeted antibodies or kinase inhibitors, respectively. Despite many successes, not all patients respond to these treatments or they develop resistance leading to cancer recurrence. The treatment of metastases remains the biggest challenge. Therefore, new therapies are required and this demands a better understanding of disease development and progression. With the two studies presented here, we aimed to gain more insight in the role of histone deacetylase 11 (HDAC11) in breast cancer, and in the function of Memo (Mediator of ErbB2-driven cell motility), in tumorigenesis and metastasis.
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\nResults from several preclinical studies suggest that the inhibition of HDACs is a promising approach to target cancer. However, the role of individual HDACs in breast cancer is largely unknown. In our study, we aimed to find HDACs with an oncogenic role in breast cancer. By screening available online databases, we discovered HDAC11 to be overexpressed in breast cancer compared to normal breast. Furthermore, we found a correlation between HDAC11 expression and ER levels in breast cancer, and could show that HDAC11 expression is induced by estrogen-signaling. Moreover, a knockdown (KD) approach revealed that HDAC11 promotes proliferation in the ER-positive breast cancer cell lines T47D and MCF7, but does not significantly alter proliferation of the ER-negative breast cancer cell lines MDA-MB-231 and SUM159. KD of HDAC11 in the ER-positive murine breast cancer cell line J110 decreased tumor growth in vivo. In addition, RNA sequencing combined with gene set enrichment analyses (GSEA) revealed that HDAC11 controls part of the estrogen responsive transcriptional program, especially genes that are normally downregulated upon estrogen induced signaling. This suggests that HDAC11 is an estrogen induced repressor of gene transcription. Furthermore, Kaplan-Meier analysis revealed that HDAC11 has prognostic value for patients who received endocrine therapy, as the probability of recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) decreased significantly for patients having tumors with high levels of HDAC11. In summary, we show that HDAC11 has oncogenic potential in ER-positive breast cancer and therefore might be a new target for therapy. 
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\nMemo was initially found through its interaction with ErbB2. Several studies have demonstrated that Memo is required for breast cancer cell migration in response to ligands activating receptor tyrosine kinases (RTKs). In this study we aimed to investigate if Memo is involved in breast cancer metastasis and tumorigenesis. Recently, we discovered that Memo is a copper-dependent redox enzyme, which promotes spontaneous metastasis from a xenograft breast cancer model. By tissue microarray analysis of primary human breast tumors we found Memo to be overexpressed in 40% of breast cancer, while expressed at low levels in the normal human breast. Moreover, high cytoplasmic Memo localization has prognostic value for early distant metastasis and death. However, primary tumor growth was not affected by Memo, neither in a xenograft model using the triple-negative human breast cancer cell line MDA-MB-231, nor in a constitutively active ErbB2 (NeuNT)-driven murine spontaneous breast cancer model. Immunohistochemistry of normal murine mammary glands revealed that Memo is expressed throughout all stages of development, specifically in the luminal cell compartment. To study the role of Memo in tumorigenesis, we generated a spontaneous NeuNT-driven murine breast cancer cell model, in which Memo excision and NeuNT expression should be achieved at the same time. Mice from all strains developed tumors with the same frequency. However, they were all positive for Memo expression, suggesting that either Memo-depleted mammary epithelial cells were outcompeted by Memo expressing cells and/or only Memo-positive cells can give rise to tumors, or that Cre-recombinase mediated excision of Memo was not functional. In summary, this study revealed that Memo is a copper-dependent redox enzyme that promotes breast cancer metastasis.
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In Vivo Evidence for the Role of CD44s in Promoting Breast Cancer Metastasis to the Liver

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

Metastasis is the leading cause of cancer-related deaths, and this occurs when tumor cells invade basement membranes and blood vessels to colonize other tissues. Metastasis is facilitated by extracellular matrix (ECM)-degrading structures called invadopodia. Recently, several microRNAs (miRNAs) have been identified as tumor and metastasis suppressor genes. Loss of expression of miR-206 has been correlated with poor prognosis in gastric cancer, breast cancer, rhabdomyosarcoma and melanoma. A previous study by others has demonstrated that restoring miR-206 expression in human breast cancer cells was sufficient to block metastasis in tumor xenograft assays. However, the targets of miR-206 that cause metastasis in these cancer models have not been fully characterized. Here, we have tested the effects of miR-206 on invadopodia in metastatic cancer cells, and identified a novel target that may explain this effect. Transducer of Cdc42-mediated Actin assembly-1 (Toca-1) is an adaptor protein that promotes formation of invadopodia and metastasis in breast cancer models, and has a predicted miR-206 binding site within the 3′ UTR of Toca-1 transcripts. To test whether the high levels of Toca-1 expression that we have observed in metastatic cancer cell lines was due to loss of miR-206, we rescued miR-206 expression using transient, stable or inducible approaches in MDA-MB-231 breast cancer, A375 melanoma, and H1299 lung cancer cell lines. In each cell model, Toca-1 expression was reduced upon miR-206 expression at both the mRNA and protein levels, thus validating Toca-1 as a new target of miR-206. Further testing of the phenotypes induced by miR-206 rescue revealed dramatic defects in ECM degradation and cell invasion in breast cancer and melanoma cell models. In subcutaneous tumor xenograft assays, stable rescue of miR-206 in H1299 tumors led to defects in tumor growth compared to those expressing a scrambled control miRNA. In addition, miR-206 expression caused a dramatic reduction in the numbers of lung metastases. We are currently extending this to our melanoma models and inducible models to define changes in metastasis-related targets of miR-206 that are aberrantly expressed in metastatic cancers. In conclusion, our study demonstrates for the first time a link between miR-206 and suppression of invadopodia formation via Toca-1 silencing, which likely contributes to the metastasis suppressing activity of miR-206 in multiple cancer types. Citation Format: Kathleen D. Watt, Peter Truesdell, Andrew W. Craig. Elucidating molecular mechanisms linking microRNA-206 loss to tumor progression and metastasis. [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 3106. doi:10.1158/1538-7445.AM2015-3106

Our studies to date have demonstrated that retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo, associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator (“suicide”) gene therapy in a variety of cancer models. Clinical testing of RRV-mediated gene therapy using the yeast cytosine deaminase (CD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug, 5-fluorouracil (5-FU), is currently underway in multi-center clinical trials being conducted in the United States for patients with recurrent high-grade glioma. In the present study, our goal has been to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers to the CNS, which occur 5-10 times more frequently than primary brain tumors. Moreover, brain metastases are often accompanied by neurological deficits, which deprive patients of their quality of life. In particular, brain-metastatic breast cancer frequently arises from highly aggressive, treatment-refractory, ‘triple-negative' (ER(-), PR(-), HER2(-)) cells, is associated with a dismal prognosis of 4-6 months, and as treatment options are largely palliative, is the main cause of death in half of these patients. Hence this condition represents an unmet medical need. We first examined the replication kinetics of RRV expressing the GFP reporter gene (AC3-emd) in human and murine breast cancer cell lines by flow cytometry. The replication kinetics of AC3-emd in the triple-negative human breast cancer cell line MDA-MB-231-BR, and in the murine breast cancer cell line JC, showed robust replication activity over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity by MTS assay after 5-FC treatment of 231BR and JC cells that had been fully transduced with RRV expressing the CD prodrug activator gene (clinical-grade vector AC3-yCD2, also designated “Toca 511”). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% even after exposure to 0.1 mM 5-FC treatment, and complete cell killing was observed after 6 days (231-BR) or 4 days (JC) exposure to 1 mM 5-FC treatment. We then examined in vivo replication kinetics of AC3-emd in intracranial 231-BR xenograft models. Intratumoral injection of 10e6 AC3-emd on Day 7 after tumor implantation was confirmed to achieve >80% transduction efficiency by flow cytometry on Day 10 post-injection. In survival studies, animals treated with AC3-yCD2+5-FC prodrug activator gene therapy showed statistically significant (p<0.0001) survival benefit compared to the AC3-yCD2+PBS control group. These data provide preclinical validation for a new Phase I dose escalation trial to evaluate RRV-mediated prodrug activator gene therapy in CNS-metastatic breast cancer, as well as other types of metastatic cancers (TOCA6 trial: clinicaltrials.gov NCT02576665), planned for initiation at the University of Miami, Cleveland Clinic, and UCLA, in mid-2016. Citation Format: Akihito Inagaki, Sara Collins, Suzanne Matsuura, Noriyuki Kasahara. Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A43.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Sli2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent. Citation Format: Dinesh K. Ahirwar, Nabanita Chatterjee, Sanjay Mishra, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4561.

Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from HuHER2 mice (MMTV.f.HuHER2 obtained from Genetech). HuHER2 mice develop breast cancer spontaneously and with a higher frequency than normal mice. We extracted primary mammary gland tumors, purified the epithelial breast cancer cells, and established 7 different HuHER2 cell lines. We also established 2 different cell lines from spontaneous lung metastases (HuHER2-L1 and -L2). We evaluated HuHER2 protein expression in the cell lines by western blot analysis. Cell surface receptor expression was evaluated by immunofluorescence (IF) staining. We performed qRT-PCR to assess phenotypic measures of aggressiveness and metastatic propensity. The following were evaluated: ER, PR, Twist1, Vimentin and E-Cadherin. Sensitivity to trastuzumab antibody, in vitro, was also assessed by evaluating changes in cellular metabolism via the MTT assay. HuHER2 protein was overexpressed in all of the isolated cell lines. One of the cell lines (denoted HuHER2-6) had approximately 1.5-8 times more protein expression compared to the other 6 cell lines developed. The two cell lines derived from spontaneous lung metastases showed approximately 1.5-fold greater HER2 protein expression than HuHER2-6. HuHER2-6 cell surface HER2 receptor staining by IF was similar to that of BT-474, a high HuHER2 expressing cell line. The 2 lung cell lines also showed comparable cell-surface staining. ER mRNA levels assessed by qRT-PCR were 20% and 0.05% of the level measured in normal mammary gland for the HuHER2-6 and HuHer2-L2 lines, respectively. The PR levels were also substantially lower (<0.01%) relative to normal mammary gland (MG) tissue. The HuHER2-6 line expressed 1.4 times more HuHER2/neu mRNA than BT-474. The mRNA for E-Cadherin in HuHER2-L1 and -L2 was 20% of MG tissue. The mRNA for TWIST1 and Vimentin were similarly elevated 3-fold and 1.5-fold, respectively, relative to MG. At trastuzumab concentrations ranging from 10 to 500 ug/ml, cell metabolism was decreased to 50%. These lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to establish a left cardiac ventricle injection model of widespread HER2/neu positive metastatic breast cancer to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies. Citation Format: Sunju Park, George Sgouros. Human HER2-overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer. [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 4279.

BackgroundSrc and signaling molecules downstream of Src, including signal transducer and activator of transcription 3 (Stat3) and cMyc, have been implicated in the development, maintenance and/or progression of several types of human cancers, including breast cancer. Here we report the ability of siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc to inhibit the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S, a widely used model for breast cancer research.Methodology/ResultsSrc and its downstream signaling partners were specifically targeted and knocked-down using siRNA. Changes in the growth properties of the cultured cancer cells/tumors were documented using assays that included anchorage-dependent and -independent (in soft agar) cell growth, apoptosis, and both primary and metastatic tumor growth in the mouse tumor model. siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc inhibited the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S. This knock-down resulted in reduced growth in monolayer and soft agar cultures, and a reduced ability to form primary tumors in NOD/SCID mice. In addition, direct intra-tumoral injection of siRNAs targeting these signaling molecules resulted in a substantial inhibition of tumor metastases as well as of primary tumor growth. Simultaneous knock-down of Src and Stat3, and/or Myc exhibited the greatest effects resulting in substantial inhibition of primary tumor growth and metastasis.Conclusions/SignificanceThese findings demonstrate the effectiveness of simultaneous targeting of Src and the downstream signaling partners Stat3 and/or cMyc to inhibit the growth and oncogenic properties of a human cancer cell line. This knowledge may be very useful in the development of future therapeutic approaches involving targeting of specific genes products involved in tumor growth and metastasis.

Introduction and Hypothesis: Breast cancer is a leading cause of cancer mortality in women due to the high frequency of metastatic disease, which, despite advances in therapeutic options, is still essentially incurable. The role of Myc in promoting tumorigenesis is beyond doubt, but there are contradictory reports in the literature on its role in the metastatic process. Using a Myc dominant negative termed Omomyc, we have demonstrated in various mouse models that Myc inhibition is a safe and effective therapeutic approach against several types of cancer, regardless of the tissue of origin or the driver oncogenic lesion. So far, Omomyc has only been tested in primary tumors. However, since many steps of the metastatic cascade have been reported to depend on Myc, we hypothesized that Omomyc could be extremely effective in both the prevention and treatment of metastasis too. Methods: We induced transgenic expression of Omomyc in a panel of 11 breast cancer cell lines and analyzed its effect on clonogenic capacity, proliferation, cell cycle progression, angiogenesis, migration, and invasion. To characterize the effect of Omomyc expression in vivo, we performed prevention and intervention studies in several mouse models of metastatic breast cancer: an orthotopic human cell line-derived model with surgical resection, a human cell line-derived lung colonization model, and the MMTV-PyMT transgenic model. Omomyc expression was induced at different stages of the disease, and tumor burden and metastatic spread were compared between groups at different time points. In parallel to this systemic modeling of Myc inhibition by transgenic expression of Omomyc, we are also validating the therapeutic utility of Omomyc-derived peptides as a pharmacologic approach. To this aim, we assessed the cell-penetrating capacity of the peptides in MDA-MB-231 cells by confocal microscopy and flow cytometry. To enhance its activity and to target metastases in vivo, Omomyc was conjugated with a metastasis-targeting sequence, and its efficacy compared with the one exerted by Omomyc alone in vitro. We selected the fusion peptide for in vivo studies and treated the orthotopic and lung colonization mouse models by several routes of administration. Results: Here we show that Omomyc expression has a dramatic effect on colony formation capacity in human breast cancer cell lines representative of all the molecular subtypes of the disease. In MDA-MB-231 cells, not only did it impair their proliferation but also migration, invasion, and their capacity to induce angiogenesis, key aspects of the metastatic process. We demonstrate that, in vivo, Omomyc reduces the growth of orthotopically implanted human breast cancer cells in immunocompromised mice, induces regression of established metastases after primary tumor resection, and impairs the development of lung metastases after tail vein injection. In the immunocompetent MMTV-PyMT transgenic model, Omomyc expression dramatically delays the formation and growth of mammary fat pad tumors, thereby preventing the appearance of lung metastases. When the Omomyc peptide is administered exogenously, we observe remarkable growth inhibition that recapitulates transgenic expression of Omomyc. When conjugated with a metastasis-targeting sequence, its cell-penetrating capacity is increased and causes abundant cell death in vitro. In vivo, treatment with the fusion peptide reduces growth of mammary primary tumors and lung metastases. Conclusions: We have demonstrated for the first time the applicability of Omomyc against metastasis, challenging the pre-established notion that Myc inhibition could potentiate, rather than inhibit, invasion. Finally, we have validated a metastasis-targeting fusion peptide as the first directly deliverable Omomyc-based drug for the treatment of metastatic breast cancer, providing a new therapeutic opportunity for patients suffering from this dreadful and incurable disease. Citation Format: Daniel Massó-Vallés, Marie-Eve Beaulieu, Toni Jauset, Erika Serrano, Sandra Martínez-Martín, Laia Foradada, Virginia Castillo, Sílvia Casacuberta-Serra, Mariano F. Zacarias-Fluck, Génesis Martín, Antonio Luque-García, Marta Escorihuela, Jonathan R. Whitfield, Joaquín Arribas, Laura Soucek. Targeting Myc in metastatic breast cancer by Omomyc: From proof of principle to pharmacologic approach [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B16.

Engineering the Extracellular Matrix to Model the Evolving Tumor Microenvironment.