Ureido-Substituted Benzenesulfonamides Potently Inhibit Carbonic Anhydrase IX and Show Antimetastatic Activity in a Model of Breast Cancer Metastasis

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.

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

Over 90% of breast cancer deaths are due to complications as a consequence of metastasis formation. Much progress has been made in understanding primary breast cancer formation; however, metastatic disease is still largely unexplored, poorly understood and incurable. Clearly, there is an urgent need for novel therapies with efficacious anti-metastatic activity. The different steps of the metastatic cascade are largely regulated by reciprocal interactions between cancer cells and their microenvironment. Accumulating evidence indicates that cells and mediators of the immune system can facilitate metastasis formation. To mechanistically study how immune cells and their mediators modulate breast cancer metastasis, we have recently developed a mouse model of spontaneous breast cancer metastasis that mimics the clinical course of metastatic disease in humans. The basis is the K14cre;EcadF/F;p53F/F transgenic mouse that develops breast cancer resembling human invasive lobular carcinoma. We orthotopically transplant invasive lobular carcinoma fragments from these mice into mammary glands of wild-type syngeneic mice. Once primary tumors are established, we mimic the clinical setting and perform a mastectomy. Following surgery, the mice develop clinically overt metastases in lymph nodes, lungs, liver and other distant organs. This novel mouse model of breast cancer metastasis accurately mimics each step of the metastatic cascade in humans. It provides a unique tool to further explore the biology of metastatic breast cancer with the aim to contribute to the development of more effective treatment strategies. Neutrophils make up a significant proportion of the inflammatory infiltrate in many tumors and their accumulation in breast cancer patients has been associated with metastasis formation. Also in our spontaneous breast cancer metastasis mouse model, metastasis formation is accompanied by a very pronounced accumulation of neutrophils in circulation and distant organs. Antibody-mediated depletion of neutrophils did not affect primary breast cancer outgrowth, but did result in a profound decrease in lung and lymph node metastasis. Using biological and genomic approaches, we have uncovered a novel communication network between gamma delta T cells and neutrophils that is critical for breast cancer metastasis. We are currently dissecting the mechanisms by which neutrophils facilitate breast cancer metastasis formation. Besides regulating metastatic disease, the immune system also modulates responsiveness of cancer to conventional forms of therapy. Using the K14cre;EcdF/F;p53F/F mouse mammary tumor model, we study the ability of the immune system to influence the anti-cancer efficacy of chemotherapy. We have observed that it is very important to optimally match chemotherapeutic drugs with immunomodulatory compounds. In addition, combining chemotherapy with an immunomodulatory drug can trigger a rewiring of the inflammatory tumor microenvironment resulting in immune-dependent therapy resistance. Taken together, through mechanistic understanding of the crosstalk between the immune system and cancer, we aim to contribute to the design of novel immunomodulatory strategies to fight metastatic breast cancer and to increase the efficacy of conventional anti-cancer therapies. (Supported by the Dutch Cancer Society grant 2011-5004, NWO/VIDI 91796307, AICR 11-0677 and FP7 MCA-ITN 317445) Citation Format: Seth B. Coffelt, Chris W. Doornebal, Metamia Ciampricotti, Camilla Salvagno, Kelly Kersten, Kim Vrijland, Cheei-Sing Hau, Jos Jonkers, Karin E. De Visser. Cancer-associated inflammation facilitates metastatic breast cancer and counteracts chemoresponsiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA07. doi:10.1158/1538-7445.CHTME14-IA07

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.

<div>Abstract<p>A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). <i>In vitro</i> assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, <i>POEM</i>, was further investigated in the model. <i>In situ</i> hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.</p></div>

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.

Breast cancer is the most common malignancy in women, with the development of distant metastases rendering the condition incurable. Relatively little is known about the factors governing the progression from primary tumor to metastasis, in part because of the difficulty in modeling what is a complex series of events. Detailed in this unit are descriptions of two murine models of breast cancer metastasis to bone that can be used to screen the effectiveness of new chemical entities on this disease process.

<div>Abstract<p>A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). <i>In vitro</i> assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, <i>POEM</i>, was further investigated in the model. <i>In situ</i> hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.</p></div>

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg−1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

BackgroundTumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.Methodology/Principal FindingsIn this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.Conclusions/SignificanceThe results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

Breast cancer is the second leading cause of cancer-related death among women, and distant metastasis is responsible for the death of ~90% of these patients. However, despite recent advances, the underlying mechanisms responsible for breast cancer metastasis remain elusive. A great impediment to this is the lack of appropriate orthotopic models of breast cancer metastasis to distant organs. In the present study, we established a novel orthotopic model of breast cancer metastasis to the lungs in mice, where metastatic sublines of 4T1 cells revealed enhanced metastatic propensity to the lungs. All mice (100%) developed lung metastasis upon orthotopic implantation of a metastatic subline of 4T1 cells, in contrast to 10% of mice with lung metastasis for a subline derived from primary tumors and 60% of mice with lung metastasis for parental 4T1 cells. At the molecular level, significant epithelial-mesenchymal transition was characterized in these metastatic sublines, which is likely at least partially, responsible for the enhanced metastasis. These established cell lines provide a novel platform to study the relevant molecular basis of metastasis and metastasis-specific therapeutics.

Carbonic anhydrase: chemistry, physiology, and inhibition.

Design, anticancer activity, and mechanistic evaluation of a novel class of selective human carbonic anhydrase IX inhibitors featuring a trifluorodihydroxypropanone pharmacophore.

The current lack of efficacy for any chemo- or molecular therapeutic in the treatment of brain metastases is thought to be due, in part, to the heterogeneous permeability of the blood-brain-barrier (BBB). Little is known about how heterogeneous permeability develops, or how it varies among individual metastases. Understanding the BBB's role in metastasis will be crucial to the development of new, more effective therapies. In this article, we developed the first magnetic resonance imaging-based strategy to detect and measure the volumes of BBB permeable and nonpermeable metastases and studied the development of altered BBB permeability in metastases in vivo, over time in a mouse model of breast cancer metastasis to the brain. Animals bearing human experimental brain metastases of breast cancer (231-BR cells) were imaged, using 3-dimensional balanced steady-state free precession to visualize total metastases, and contrast-enhanced T1-weighted spin echo with gadopentetic acid (Gd-DTPA) to visualize which of these displayed contrast enhancement, as Gd-DTPA leakage is indicative of altered BBB permeability. Metastases detected 20 days after injection showed no Gd-DTPA enhancement. At day 25, 6.1% ± 6.3% (mean ± standard deviation) of metastases enhanced, and by day 30, 28.1% ± 14.2% enhanced (P < 0.05). Enhancing metastases (mid: 0.14 ± 0.18 mm, late: 0.24 ± 0.32 mm) had larger volumes than nonenhancing (mid: 0.04 ± 0.04 mm, late: 0.09 ± 0.09 mm, P < 0.05); however, there was no significant difference between the growth rates of the 2. A significant number of brain metastases were uniformly nonpermeable, which highlights the need for developing treatment strategies that can overcome the permeability of the BBB. The model developed herein can provide the basis for in vivo evaluation of both BBB permeable and nonpermeable metastases response to therapy.

In previous studies using several experimental models expressing different progesterone receptor (PR) isoform ratios, we have shown that only those with high levels of isoform A (PRA) are inhibited by antiprogestins whereas those with high levels of isoform B (PRB) are resistant to antiprogestin therapy. Moreover, results obtained using tissue cultures of breast cancer patients confirmed the data observed in these experimental models (May and Rojas et al., ASCO meeting 2015). The aim of this study was to evaluate the role of progestins and antiprogestins on the outgrowth of spontaneous metastatic foci of mammary carcinomas with different PR isoform ratios. We used metastatic tumors from the murine medroxyprogesterone acetate (MPA)-induced breast cancer model: C7-2-HI (PRA&amp;gt;PRB) and C7-HI (PRA&amp;lt;PRB), and human MDA-MB-231 cells genetically modified to overexpress PRB. Tumors were orthotopically inoculated in the fourth mammary gland and Mifepristone (MFP; 6 mg), Telapristone (TLP; 6 mg), MPA (20 mg) or vehicle silastic pellets were sc implanted when the tumors reached a size of 25 mm2. Tumors were measured twice a week and animals were followed to detect the presence of axillary lymph node metastasis. Animals were euthanized before tumor size exceeded 2 cm at the largest diameter or showed a deterioration of the physical conditions. Both antiprogestins had similar effects, MFP induced almost complete regression of the C7-2-HI tumor as already described, and TLP inhibited significantly its growth as compared with control tumors, while no changes in tumor size were observed in MPA-treated mice. Lung or lymph node studies confirmed the lack of metastatic foci in MFP-treated mice while a significant decrease (p&amp;lt;0.05) and increase (p&amp;lt;0.05) was observed in TLP- and MPA-treated animals respectively, as compared to control mice. In C7-HI tumors, with higher levels of PRB than PRA, MFP clearly stimulated the number of lung foci as compared with control animals (p&amp;lt;0.05). The effects of TLP and MPA on tumor metastasis, although not inhibitory, need to be confirmed. Studies performed using MDA-MB-231-PRB xenografts growing in NSG mice showed that MFP stimulated the growth of lung foci, while MPA was inhibitory. No significant changes were observed in mice inoculated with empty vector transfected cells. To investigate the mechanisms underlying the metastatic process, we evaluated the expression of a metastatic suppressor gene (Nm23-H1) and a prometastatic enzyme (MMP-2). We found a lower expression by immunohistochemistry of the former and an increase in the latter by qPCR in MFP-treated MDA-MB-231-PRB xenografts compared with untreated tumors. In conclusion, we have demonstrated that MFP impedes the metastatic process in tumors with higher levels of PRA than PRB while it might promote metastasis in those with the opposite ratio. We suggest that MFP, through PRB receptors, downregulate the expression of Nm23-H1 and increase the expression of MMP-2. The conclusive effects of TLP and MPA need further investigation. These data underscore the importance of isoform ratio determination before treatment of breast cancer patients with endocrine therapies that target PR. Citation Format: Lanari C, Alvarez M, Giulianelli S, Marina R, Sequeira G, May M, Novaro V, Sahores A, Lamb C. The ratio of progesterone receptor isoform A to B determines the effect of antiprogestins on preclinical models of breast cancer metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-27.