Metastatic 4T1 Cells Research Articles

Abstract 1555: MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis

Abstract Metastatic breast cancer is the second leading cause of cancer-related death among women. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El-Andaloussi, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Mhmet F. Demirci, Gang Zhou. MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis. [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 1555.

Abstract 717: Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model

Abstract It is widely accepted that the epithelial-mesenchymal plasticity of malignant cells is required during cancer metastatic cascade. The complex phenotypic changes highly depend on the collaboration of various molecular signaling and extracellular cues originating from wide range of stromal cells in the tumor microenvironment. However, the specific mechanisms of how EMT plasticity spatiotemporally regulates metastasis are poorly defined. Myeloid-derived suppressor cells (MDSCs) have been identified in most cancer patients and animal models due to their immune suppressive functions, but recent studies implicate their direct role in promoting metastasis by activating tumor-angiogenesis. To determine the roles of MDSCs in breast cancer metastasis, we utilized murine breast cancer cells, non-metastatic EMT6 and metastatic 4T1 cells. We showed that the metastatic 4T1 murine breast tumors induced early systemic expansion and mobilization of MDSCs in distant sites as well as in the primary tumor. We investigated the direct functions of MDSCs in tumor progression by isolating monocytic and granulocytic MDSCs from primary tumor, lung and bone marrow of tumor-bearing mice and then they were co-cultured with non-metastatic EMT6 cells. We found that tumor infiltrating m-MDSCs from 4T1 tumor-bearing mice increased the expression of Vimentin, Twist1, TGF-â and IL-6 in EMT6 tumor cells. In contrast, flow cytometry sorted lung infiltrating MDSCs from 4T1 tumor-bearing mice enhanced the EpCAM expression and proliferation in EMT6 cells. Cell invasion assay showed that invasive ability of EMT6 cells were significantly increased when they were co-cultured with m-MDSCs while g-MDSCs slightly decreased the number of invaded cells, compared to control group. We utilized immunofluorescence staining and confirmed the increased expression of Vimentin, CK14 (cytokeratin 14) in EMT6 cells co-cultured with m-MDSCs. In contrast, g-MDSCs induced down-regulation of these markers while they increased cell proliferation as assessed by Ki67 staining. Furthermore, flow cytometry analysis showed the increased CD24+CD29+ population, a marker of murine cancer stem cell (CSC) phenotype, in EMT6 cells when co-cultured with m-MDSCs from 4T1 tumor-bearing mice. Tumor sphere assay confirmed that m-MDSCs enhanced sphere forming ability of tumor cells. Taken together, these data suggest that m-MDSCs derived from metastatic 4T1 tumor-bearing mice are able to confer EMT/CSC phenotype on tumor cells while g-MDSCs are more potent in inducing epithelial phenotype and proliferation in tumor cells. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Mehmet Demirci, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Gang Zhou, Hasan Korkaya. Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model. [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 717.

In Vivo Capture of Circulating Tumor Cells Based on Transfusion with a Vein Indwelling Needle.

Detection of circulating tumor cells (CTCs) could be used as a "liquid biopsy" for tracking the spread of cancer. In vitro detection methods based on blood sampling and in vitro CTC capture often suffer from the small sampling volume and sampling error. Here, the in vivo capture of CTCs based on transfusion with a surface-modified vein indwelling needle is proposed. When the needle was applied to transfusion in the vein, the simultaneous capture of CTCs was performed. To investigate the actual capture efficiency of the in vivo capture method, labeled MCF-7 cells were directly injected into the veins of rabbits, wild type mice, and nude mice and could be successfully captured. Two of 5 MCF-7 cells injected into the veins of nude mice were successfully captured. To investigate the CTC capture of mouse tumor model and compare with the in vitro method, mice were subcutaneous inoculated with metastatic 4T1 cells. Seven and 21 days after inoculation, CTCs were captured for the first time using in vivo and in vitro methods, respectively. This predicted that the in vivo method could be more suitable for use of early diagnosis of cancer than the in vitro method. As CTC capture can be performed at the same time as transfusion and does not cause further bodily harm, it would be easily accepted by patients. This efficient, simple, and less damaging method involving the use of a vein indwelling needle could be popularized easily in the clinic.

Unraveling the Role of Huntingtin in Breast Cancer Metastasis

Huntingtin (HTT) is mutated in Huntington's disease but is ubiquitously expressed, and mutant HTT influences cancer progression. We investigated wild-type HTT function during breast cancer. We analyzed HTT and ZO1 expression as well as the HTT phosphoserine 421-activated form (S421-P-HTT) in human breast cancer tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We performed in vitro migration and invasion assays as well as in vivo tail vein injections of the metastatic 4T1 cells in BALB/c mice (n = 11 per group). We analyzed tumor progression in knock-in mice with modified S421 crossed with the MMTV-PyVT mammary cancer model (at least n = 12 per group). Data were analyzed with unpaired t tests, analysis of variance, Pearson or Spearman correlation, and Mann Whitney or Kruskal-Wallis tests. All statistical tests were two-sided. Levels of HTT and of S421-P-HTT are abnormally low in poorly differentiated and metastatic human breast cancers. HTT expression is downregulated in invasive compared with in situ carcinoma (P < .001). In BALB/c mice, silencing of HTT promotes lung colonization by a metastatic mammary cancer cell line (P = .005) and S421-unphosphorylatable-HTT accelerates cancer progression. HTT interacts with ZO1 and regulates both its expression and its localization to tight junctions. In human breast tumors, the patterns of HTT and ZO1 expression are similar (Pearson correlation coefficient = 0.66, P < .001). HTT may inhibit breast tumor dissemination through maintenance of ZO1 at tight junctions. Downregulation of HTT transcript and protein levels is a prognostic factor for poor prognosis and metastasis development.

Abstract 466: Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model

Abstract Metastatic breast cancer is the second leading cause of cancer-related death among women in the United States. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Abdeljabar El Andaloussi, Ena Novakovic, Raziye Piranlioglu, Mehmet F. Demirci, Shawn G. Clouthier, Max S. Wicha. Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model. [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 466. doi:10.1158/1538-7445.AM2015-466

Abstract 154: Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis

Abstract Background: Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. TGFβ signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Methods: Genome-wide lncRNA profile was performed using NCode™ Mouse Noncoding Microarray chip from Life Technologies, which contains 10,802 lncRNAs and 25,178 protein-coding genes. Total RNA was isolated from mouse mammary epithelial NMuMG cells, which had been treated with TGFβ for 0, 12 and 24 hours, and was used to hybridize the microarray. The role of a top TGFβ-upregulated lncRNA, lncRNA-HIT, in EMT and metastasis was further characterized in cell culture and orthotopic breast cancer models Results: Among 10,802 lnRNAs profiled, 680 were up-regulated and 633 were down-regulated during TGFβ-induced EMT, respectively. Further, we identify that lncRNA-HIT (HOXA antisense transcript induced by TGFβ) mediates TGFβ function, i.e., depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth and metastasis. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Conclusions: These data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers. Citation Format: Edward Richards, Gu Zhang, Jennifer Permuth-Wey, Zhu-Peng Li, Sridevi Challa, William Kong, Dan Su, Domenico Coppola, Wei-min Mao, Thomas Sellers, Jin Q. Cheng. Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer 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 154. doi:10.1158/1538-7445.AM2015-154

P026 Mechanical ventilation promotes lung metastasis in 4T1 breast cancer lung-metastasized models

Goals: Recent evidence suggests that general anaesthesia and perioperative factors significantly increased risk of cancer recurrence compared to locoregional anaesthesia in patients undergoing surgery, however, their effects on behavior of cancer cells are not yet known. The endothelial cell and epithelial disruption was observed during mechanical ventilation, general anaesthesia in animalmodels. This studywas to test the hypothesis thatmechanical ventilation during cancer surgery induces lung stroma/tissue milieu changes, creating a favorable microenvironment for postoperative lung metastatic tumor establishment. Methods: Female BALB/c mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =8) and a control group (no ventilation; n =8). All the mice were subjected to intravenous injection of murine mammary adenocarcinoma 4T1-green fluorescent protein (GFP) 10 cells during anaesthesia. 24 hr later, sections from the lung tissue were examined and 4T1 celler were counted by direct GFP fluorescence observation, hematoxylineosin histology and Ep-CAM immunohistochemistry. To closely mimic human cancer surgical and anaesthetic manipulation, A clinically relevant animal model of spontaneous breast cancer lung metastasis with surgical resection of primary tumor was used to investigate the purified mechanical ventilation event that dictate lung metastasis post-operation. Female BALB/c mice were inoculated with metastatic murine mammary adenocarcinoma 4T1 cells in the mammary fat pad (3×105/mouse). After 14 day growth, mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =6) and a control group (no ventilation; n =6). All the mice were subjected to flank tumor resection during anaesthesia. Two weeks later, Metastatic tumor burden was assessed by both macroscopic metastatic nodule count and hematoxylin-eosin histology. Results: Mechanical ventilation was associated with increased circulating breast tumor cells arrest at microvasculature of lung (P = 0.01). The histology analysis data indicated that mechanical ventilation induced endothelial-epithelium abnormalities, inflammatory reaction and significantly upregulated the expression of epithelial adhesion molecules (Ep-CAM). Postoperative metastases exhibited significantly increase in mechanical ventilated groups in comparison with those in non-ventilated group (P < 0.05). Conclusion: Mechanical ventilation led to a progressive phenotype of lung metastases that exhibited a microenvironment for cancer cell adhesion, invasion, survive and growth within lungs. Disclosure of Interest: No significant relationships. P027 The analysis of cytogenetic intra-tumoral heterogeneity in squamous cell carcinoma of the breast M. Oikawa *, A. Igawa, M. Ishida, Y. Nakamura, S. Nishimura, C. Koga, S. Akiyoshi, Y. Koi, K. Taguchi, S. Ohno. Breast Oncology, National Kyushu Cancer Center, Fukuoka, Japan, Pathology, National Kyushu Cancer Center, Fukuoka, Japan

Long Non-coding RNAs (LncRNA) Regulated by Transforming Growth Factor (TGF) β: LncRNA-HIT-MEDIATED TGFβ-INDUCED EPITHELIAL TO MESENCHYMAL TRANSITION IN MAMMARY EPITHELIA

Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Analysis of the in vivo and in vitro effects of photodynamic therapy on breast cancer by using a sensitizer, sinoporphyrin sodium.

Photodynamic therapy (PDT) is an emerging theranostic modality for various cancers and diseases. Photosensitizers are critical components for PDT. Sinoporphyrin sodium, referred to as DVDMS, is a newly identified photosensitizer that was isolated from Photofrin. Here, we evaluated the effects of DVDMS-mediated PDT (DVDMS-PDT) on tumor cell proliferation and metastasis in the highly metastatic 4T1 cell line and a mouse xenograft model. DVDMS-PDT elicited a potent phototoxic effect in vitro, which was abolished using the reactive oxygen species (ROS) scavenger N-acetylcysteine. In addition, DVDMS-PDT effectively inhibited the migration of 4T1 cells in scratch wound-healing and transwell assays. Using an in vivo mouse model, DVDMS-PDT greatly prolonged the survival time of tumor-bearing mice and inhibited tumor growth and lung metastasis, consistent with in vitro findings. PDT with DVDMS had a greater anti-tumor efficacy than clinically used Photofrin. Moreover, preliminary toxicological results indicate that DVDMS is relatively safe. These results suggest that DVDMS is a promising sensitizer that warrants further development for use in cancer treatment with PDT or other sensitizing agent-based therapies.

Abstract 592: HOXD10 suppresses mammary tumor growth and metastasis coincident with decreased CD11b+Gr1+ leukocyte infiltration and angiogenesis

Abstract The tumor suppressor gene HOXD10 is frequently lost in breast cancer, and we have previously shown that restoring expression of this master transcriptional regulator can revert mammary tumor cells to a normal, polarized phenotype and inhibit their growth in nude mice (Carrio et al., Cancer Res., 2005). In addition, we showed that restoring HOXD10 also reduced VEGFA expression and decreased endothelial cell migration in vitro (Chen et al., Cancer Res., 2009). Considering the critical role of the immune system in mammary tumor progression and metastasis, we investigated whether Hoxd10 could also prevent breast tumor progression in an immunocompetent model. Metastatic 4T1 cell lines were transduced with Hoxd10 and were either orthotopically or intravenously injected into four to eight week old, female Balb/c mice. We observed a significant reduction in tumor burden at both primary at metastatic sites in tumors expressing Hoxd10. Immunohistochemical staining for CD31 of primary tumors expressing Hoxd10 revealed decreased angiogenesis. Flow cytometry and immunohistochemical staining also revealed a decrease in infiltrating Cd11b+Gr1+ leukocytes in Hoxd10-expressing tissues. Gene and protein analysis revealed that in addition to decreased VEGFA, HOXD10 also reduced expression of CCL2, a key chemokine linked to recruitment of Cd11B+Gr1+ leukocytes. Using an in vitro migration assay, we confirmed that the HOXD10-mediated reduction in CCL2 expression was directly linked to reduced recruitment of monocytes to tumor cells. Together, these data show that HOXD10 can stabilize the tumor microenvironment by coordinately reducing tumor cell growth, angiogenesis, leukocyte recruitment, and metastasis, and suggest a novel and comprehensive therapeutic approach to treat mammary tumors. Citation Format: Suraj Kachgal, Amy Chen, Mazen Sidani, Shahrzad Afghani, Nancy J. Boudreau. HOXD10 suppresses mammary tumor growth and metastasis coincident with decreased CD11b+Gr1+ leukocyte infiltration and angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 592. doi:10.1158/1538-7445.AM2014-592

Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling

Most deaths associated with breast cancer, the most common malignancy in women, are caused by metastasis. Tumor associated macrophages significantly contribute to breast cancer progression and development of metastasis through the promotion of angiogenesis which involves a central regulator of macrophage functions: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Macrophages are activated by macrophage colony stimulating factor (MCSF) and chemokine (C-C motif) ligand 2 (CCL2) to secrete angiogenic factors including vascular endothelial growth factor (VEGF). The release of MCSF from tumor cells is mediated by ectodomain shedding through tumor necrosis factor alpha converting enzyme activation (TACE). Here we determined whether tumor cells TACE-shed MCSF promotes angiogenesis through activation of the NF-κB pathway in macrophages and the subsequent release of VEGF. These interactions were modeled in vitro using a panel of mammary cells mimicking the breast cancer progression from normal murine mammary gland cells to metastatic 4T1 cells along with J774 macrophages, all derived from BALB/c mice. TACE and MCSF expressions were higher in metastatic cells compared to epithelial cells (p < 0.05). Tumor conditioned medias activated the expression of VEGF by macrophages through stimulation of the NF-κB pathway and resulting macrophage secretions that promoted high levels of endothelial cell tubes. Furthermore, the combinations of CCL2, also highly expressed by tumor cells, and MCSF promoted pro-angiogenic macrophages. These results highlight the key role of tumor cell TACE-shed MCSF and secreted CCL2 in stimulating pro-angiogenic macrophages.

Abstract B48: Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling

Abstract Mortalities associated with breast cancer, the most common malignancy in women, are mainly due to metastases. Tumor associated macrophages (TAMs) significantly contribute to the development of metastasis through promotion of the rate limiting step of angiogenesis through secretion of vascular endothelial growth factor (VEGF). Macrophage differentiation and migration are activated by macrophage colony stimulating factor (MCSF) and chemokine (C-C motif) ligand 2 (CCL2), respectively. Whereas tumor cells secrete CCL2, MCSF release is mediated by ectodomain shedding through tumor necrosis factor alpha converting enzyme activation (TACE). How tumor cells support macrophage promotion of angiogenesis remains unclear. In the present study, we determined whether MCSF shed by TACE from tumor cells promotes angiogenesis through the activation of NF-κB signaling in macrophages and subsequent VEGF release. These studies were modeled in vitro using a panel of murine mammary cells mimicking breast cancer progression from normal murine mammary gland (NMuMG) cells to metastatic 4T1 cells along with J774 macrophages. TACE and MCSF expressions were higher in metastatic cells compared to epithelial cells (p&amp;lt;0.05). After incubation with 4T1 tumor conditioned media (CM), macrophages secreted significantly more VEGF and expressed of the activated NF-κB, and the resulting macrophage secretions promoted significantly high levels of endothelial cell tubes (p&amp;lt;0.05). Furthermore, a similar activation of macrophages was generated by the combination of CCL2, also highly expressed by tumor cells, and MCSF. These results highlight the key role of tumor cell TACE-shed MCSF and secreted CCL2 in synergistically stimulating pro-angiogenic macrophages. Citation Format: Stephen L. Rego, Rachel Helms, Didier Dreau. Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B48.

Abstract B065: The RSK/YB-1 pathway represents an opportunity for targeting TNBC and holds promise of treating metastases

Abstract Triple-negative breast cancers (TNBC) account for 15-25% of all breast cancers, have poor outcomes and high rates of relapse. Along with metastatic disease treatment options for TNBC are limited to that of conventional chemotherapy. The lack of targeted therapies for these cancers is a distinct unmet clinical need. YB-1, a transcription factor, is associated with 70% of TNBC and poor prognosis. While YB-1 is not easily druggable, p90 ribosomal S6 kinase (RSK), which lies upstream of YB-1 and activates it by phosphorylation of serine 102, is an ideal candidate. We recently reported that RSK inhibition decreases TNBC cell growth. Also RSK and YB-1 are implicated in invasion and therefore may play a role in metastatic spread. Herein, we asked whether RSK inhibitors would be beneficial for patients with metastatic disease. Our concern for treating metastatic disease was inspired by a study we conducted in 2222 patients with breast cancer. Women who had local, regional or distant metastases were at a much higher risk of dying. Remarkably those with distant metastases were 100 times for likely to die from breast cancer as compared to those without disseminated disease. Further, women with TNBC specifically had the worst outcomes and their time to death was the shortest of any breast cancer subtype. We therefore conducted a screen of 128 compounds, which are in clinical trials, in SUM149 TNBC cells and compared them to the RSK inhibitor BI-D1870. Most of these drugs failed to inhibit TNBC growth; however, BI-D1870 was highly active. These promising results point towards RSK as a potential molecular target for TNBC yet there are no inhibitors available for use in patients at this time. To further validate RSK as a target for TNBC we asked which of the four RSK isoforms (RSK1-4) are expressed. Only RSK 1 and 2 are expressed in breast cancer cell lines. Inhibiting RSK by siRNA or BI-D1870 suppressed the growth of TNBC cells by ~90%; however, there was less of an effect on non-TNBC cells where growth was attenuated by ~50%. RSK inhibition with siRNA also triggered apoptosis to a greater degree in TNBC cell lines. There was no growth inhibitory effect on normal mammary epithelial cells (184htrt). Further, RSK inhibition suppressed the growth of TNBC colonies in soft agar by ~90%. Kinexus antibody arrays were used to understand why loss of RSK suppressed the growth of TNBC. Of note, cell proliferation, invasion and apoptosis proteins were suppressed indicating the multifaceted benefit of inhibiting RSK. Next we asked whether the RSK/YB-1 pathway was active in metastases. We assessed activated RSK in a panel of metastatic murine breast cancer cell lines. The RSK pathway was more active in the metastatic cell lines compared to the non-metastatic cells. Taking this further, we compared the non-metastatic 67NR to the metastatic 4T1 cells where RSK 1 and 2 were more highly expressed in the latter. Likewise, P-YB-1S102 was more active. Transfecting 67NR cells with activated RSK1 increased their invasion. Conversely inhibiting RSK with BI-D1870 decreased the growth and invasion of the 4T1 cells. In a mouse xenograft model, P-YB-1S102 was highly expressed in the lung and liver metastases obtained from the 4T1 cells. Similarly, P-YB-1S102 was active in a distant metastases obtained from a patient with TNBC. Subsequently, we identified three RSK inhibitors from a chemical library screen. The most active agent, compound 2, had an in vitro IC50=10nM which was comparable to BI-D1870. Accordingly compound 2 inhibited TNBC growth and signaling through YB-1. In conclusion we identify RSK as a promising therapeutic target for TNBC that has the potential to suppress the growth of metastases. Citation Format: Anna L. Stratford, Rachel Berns, Pauline So, Mary R. Pambid, Fotovati Abbas, Samah Abu-Ali, Kaiji Hu, Kevin Bennewith, Edie Dullaghan, Sandra E. Dunn. The RSK/YB-1 pathway represents an opportunity for targeting TNBC and holds promise of treating metastases. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B065.

Sugar and Heavy Atom Effects of Glycoconjugated Chlorin Palladium Complex on Photocytotoxicity

Palladium(II) complexes of glycoconjugated porphyrin and pyrrolidine-fused chlorin were prepared to examine sugar and heavy atom effects on in vitro photocytotoxicity. Cellular uptake into HeLa cells was enhanced by introducing sugar units regardless of other features, such as the central ion (free base or palladium(II) ion) and the ring structure (porphyrin or chlorin). The palladium(II) complex of glycoconjugated pyrrolidine-fused chlorin (PdPC2) exerted an excellent degree of photocytotoxicity not only on HeLa cells, but also on metastatic B16-BL6 cells, weakly metastatic B16F1 cells, and metastatic 4T1 cells. However, free-base glycoconjugated pyrrolidine-fused chlorin (PC2) also exerted similar or much higher photocytotoxicity rather than PdPC2. Therefore, the palladium(II) ion did not improve the in vitro photocytotoxicity of PC2. The enhanced singlet oxygen generation of palladium(II) complexes (i.e., the heavy atom effect) was confirmed at least in O(2)-saturated D(2)O. In addition, the formation of hydrogen peroxide and hydroxyl radical were also detected in O(2)-saturated phosphate buffered saline. However, the reactive oxygen species (ROS) generation efficiency, which is the product of the (relative) quantum yield of each ROS and the light absorbing ability, did not fit the trends of photocytotoxicity seen for the photosensitizers. In our glycoconjugated photosensitizers tested, the best indicator of the photocytotoxicity was found to be the light absorbing ability (namely, the oscillator strength in the wavelength region applied in the photocytotoxicity test). These results indicated that photochemical characteristics of glycoconjugated photosensitizers were notably susceptible to the microenvironment. The biological characteristics, such as the sugar effect, were a much more reliable approach to improving the photocytotoxicity of photosensitizers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Myeloid Suppressor Cells Regulate the Lung Environment—Letter

4T1 murine mammary carcinoma cells implanted in syngeneic Balb/c mice are increasingly being used in metastasis research, with some groups using this model to study tumor-induced accumulation of bone marrow-derived cells in metastatic target organs. Bone marrow-derived cells (including CD11b(+)Gr-1(+) myelomonocytic cells) are thought to modify the local lung microenvironment to facilitate subsequent colonization by metastatic tumor cells. While quantification of metastatic 4T1 tumor cells in various tissues can be done using ex vivo colony-forming assays, detection of metastatic 4T1 cells is often facilitated by expressing fluorescent proteins in the tumor cells prior to implantation. We found that Balb/c mice mount a potent immune response against 4T1 cells expressing green fluorescent protein (GFP) that includes the generation of anti-GFP antibodies in the circulation. Importantly, the number of bone marrow-derived CD11b(+)Gr-1(+) cells and metastatic tumor cells that accumulate in the lungs is significantly decreased in mice implanted with 4T1 cells expressing GFP compared with mice bearing wild-type 4T1 tumors. Taken together, our data caution against the use of GFP-expressing tumor cells in the Balb/c mouse strain, particularly for studying the influence of immunomodulatory cells on tumor cell metastasis.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Abstract P6-15-11: Role of Proteasome Activators in the Inhibition Breast Tumors and Suppression of Metastasis

Abstract Background: Breast cancer is the second leading cause of cancer-related death of women in the U.S. A number of genes involved in the process of growth, proliferation, apoptosis and DNA repair have aberrant expression in tumor cells. Metastasis is major cause of the decline in the quality of life breast cancer patients. Heat shock protein, Hsp25 is known to participate in tumorigenesis, and is highly expressed in breast tumors biopsies. However, the exact role of Hsp25 is incompletely understood. Materials and Methods: We have silenced the expression of Hsp25 using lentiviral constructs of short interfering RNA in highly metastatic mouse 4T1 cell line. Mouse tumor formation and metastasis assay was done by subcutaneous injection of 4T1-controlshRNA and 4T1-Hsp25shRNA cells into the mammary pad of female BALB/c mice. Tumor progression/regression was monitored by using an in vivo animal imaging system (MaesteroTM In-vivo imaging system, CRI, Woburn, MA, USA). Microarray analysis was done by using protocols of Applied microarray, Inc, USA. In vivo antitumor assay and adoptive transfer of CD8+ T cells was done by using standard protocol. Results: Microarray investigation on Hsp25 silenced cells demonstrated an upregulation of tumor suppressor gene (N-myc downstream regulated gene 2) and down regulation of metastasis gene (urokinase-type plasminogen activator) signatures. Subcutaneous injection of Hsp25 silenced 4T1 tumor (4T1-Hsp25shRNA) cells into immunocompetent female BALB/c mice results in complete tumor regression, absence of pulmonary metastasis and enhanced CD8+ CTL-dependent tumor killing. In vivo depletion of CD8+ T cells allowed the 4T1-Hsp25shRNA tumors to grow; however, there was no observable metastasis to the lungs. These results were confirmed by in vitro cytotoxicity assays in which CD8+ T cells recovered from mice injected with 4T1 -Hsp25shRNA cells, but not 4T1-controlshRNA cells effectively lysed 4T1 targets. Furthermore, the adoptive transfer of 4T1-Hsp25shRNA reactive CD8+ T cells into mice injected with 4T1-controlshRNA cells enabled them to effectively reject 4T1 tumors. Discussion: Our study strongly indicate Hsp25 as a therapeutic target for breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-11.

Abstract S6-8: Hedgehog Ligand Overexpression Predicts Poor Outcome in Breast Cancer and Is a Potential Therapeutic Target for Metastatic Disease

Abstract Background: The Hh signalling pathway plays an important role in a number of malignancies, and accumulating data suggest it contributes significantly to the development and progression of breast cancer. However, there is scant data regarding the clinical significance of its dysregulation in breast cancer and its functional effects in breast cancer models. Methods: We investigated the clinical significance of the expression of Hh pathway components using immunohistochemistry in a well-characterised cohort of 279 patients with invasive ductal carcinoma (IDC) to determine its prognostic significance. A murine mammary cancer allograft model based on the M6 cell line, derived from the C3/SV40T transgenic mouse was used to determine the effects of Shh overexpression on tumour growth in vitro and in vivo. Finally, the therapeutic potential of Hh blockade using the monoclonal antibody 5E1 was also explored in two mouse mammary carcinoma models; the M6 allograft model and the highly metastatic 4T1 cell line in immunocompetent Balb/C mice. Results: High Hh ligand expression was observed in 34% of IDC and was associated with increased risk of breast cancer recurrence (HR 1.95, p=0.0004) and breast cancer specific death (HR 2.3, p=0.0002). High Hh expression was also associated with the basal-like subtype (p=0.004). Overexpression of Hh ligand in M6 cells transplanted to fat pads of immunodeficient mice resulted in a 4 fold increase in tumor volume (p= 0.006) and was associated with upregulation of the canonical Hh target genes Gli1 and HHIP. However, Shh promoted peritumoral lymphatic invasion in 4/5 mice compared to only 1/5 in the vector control group and was associated with a significantly shorter time to the development of metastatic disease (p=0.0004). M6 cells overexpressing Hh ligand showed no change in proliferation, self renewal or migration in vitro, suggesting a requirement for stromal interaction in Hh-dependent tumor promotion. Finally, we found that the enhanced tumor growth conferred by Hh ligand overexpression could be blocked by administration of a neutralizing antibody to Hh ligand. Furthermore, using a metastatic mammary carcinoma isograft model (4T1) that endogenously expresses Hh ligand, we demonstrate that Hh blockade resulted in smaller lung metastatic deposits (p=0.045). Conclusions: We found that high Hh ligand is associated with a poor prognosis in IDC. Overexpression of Hh ligand promotes murine mammary tumor growth, which requires stromal interaction, as well as lymphatic vessel invasion. Finally, we demonstrate that blockade of the Hh ligand is a potential therapy in metastatic breast carcinoma. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-8.

Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis

AbstractThe coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis.