Metastatic Breast Cancer Growth Research Articles

TMIC-68. SIRPΑ BLOCKADE AS AN IMMUNOTHERAPEUTIC STRATEGY TO TREAT BREAST CANCER BRAIN METASTASIS

Abstract Triple-negative breast cancer (TNBC) has a high incidence of brain metastasis, with no standard therapeutic strategy for treatment. Therefore, new targets are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, direct targeting SIRPα as a potential approach to mitigate metastatic burden remains understudied. Single-cell-RNA-sequencing indicates that SIRPα expression on basal epithelial cells, and TCGA data shows SIRPα expression is associated with a significant reduction in survival in basal-like breast cancer. Our immune staining against SIRPα in breast cancer patient biopsies shows a 3.5-fold increase in expression in metastatic lesions compared to the primary tumor (n=19; p ≤ 0.01). Staining of 4T1 parental and brain-trophic 4T1-Br3 cells showed an 84% increase in SIRPα in the metastatic cells (n=3; p ≤ 0.05). Furthermore, Real-time cell impedance analysis revealed that SIRPα blockade inhibits TNBC 4T1br3 cell migration (n=4; p≤0.01), suggesting a link between SIRPα expression and metastatic potential. Furthermore, in vivo SIRPα blockade reduced brain metastatic burden by approximately 90%, (n=4-7; p ≤ 0.05). Digital spatial profiling of tissues revealed that SIRPα blockade was associated with reductions in immune checkpoints, including CTLA-4, PD-1, PD-L1, and LAG3, and immunosuppressive T-cell/monocyte populations. Furthermore, our spatial analysis indicated that brain lesions of WT mice have increases in the extracellular matrix protein fibronectin in the immune enriched regions, potentially serving as a barrier for immune cell infiltration contributing to invasion, metastasis, and immune evasion, was reduced by 70% in SIRPα treated brain lesions (n-3-6; p ≤ 0.05). Thus, these data suggest that SIRPα blockade may influence the tumor microenvironment to limit brain metastatic breast cancer growth and potentially enhance patient survival to metastatic brain disease.

Abstract P1-14-04: A Phase 0 Clinical trial of Sacituzumab Govitecan in Patients with Breast Cancer Brain Metastases and Recurrent Glioblastoma

Abstract Purpose: Sacituzumab govitecan has shown efficacy and acceptable tolerability in a multicenter phase I/II clinical trial (NCT01631552) in patients with advanced epithelial cancers. Our study was initiated to determine the bioavailability of Sacituzumab govitecan (SG) in breast brain metastasis and glioblastoma. The goals were to evaluate the extent by which SG can penetrate the blood brain barrier and access tumor tissues by testing free SN-38, SN-38G and total SN-38 concentrations in tumor tissue, serum, and CSF. Patients and methods: Patients diagnosed with brain metastatic breast cancer and recurrent glioblastoma were enrolled in a single-center clinical phase 0 study to receive a single intravenous dose of SG at 10 mg/kg one day before surgical resection. Tumor and corresponding serum were collected during surgery to measure their levels of SN-38 and its metabolites. Following recovery, patients resumed SG treatment at 10 mg/kg on days 1 and 8 of 21-day cycles and were assessed for responses by MRI every third cycle using response assessment in neuro-oncology (RANO) criteria. Total Sn-38 levels were quantified in tumor tissue and corresponding serum from the patients. Trop-2 and carbonic anhydrase IX (CAIX) expression was investigated by IHC. SG activity was tested in a breast cancer intracranial mouse model. Results: An average of 2365 ng/ml in serum and 132 ng/g in tissue of total SN-38 was quantified in our patient samples. Trop-2 expression was observed in 90% of patient tumors. 40% of the samples showed high expression of CAIX. SG significantly inhibited tumor growth in vivo and increased overall survival. 20% of patients in the breast cancer arm of the trial demonstrated a complete response by RANO criteria and the remaining 80% of patients demonstrated a partial response. Ultimately, 80% of the patients in the breast cancer metastasis arm survived. Conclusion: Sacituzumab govitecan is an effective drug that crosses the blood brain barrier to inhibit the growth of metastatic breast cancer to the brain after tumor resection surgery. Citation Format: Pegah Ghamasaee, Henriette Balinda, Andrew Brenner, John Floyd. A Phase 0 Clinical trial of Sacituzumab Govitecan in Patients with Breast Cancer Brain Metastases and Recurrent Glioblastoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-14-04.

Nanoparticle STING Agonist Reprograms the Bone Marrow to an Antitumor Phenotype and Protects Against Bone Destruction.

Bone metastases are difficult to treat due to the inaccessibility of the bone marrow compartment and the immunosuppressive microenvironment that protects resident stem cells. Packaging a STING agonist into a nanoparticle that enables systemic administration and drug accumulation at tumor sites overcomes both barriers to stymie metastatic breast cancer growth.

TAMI-05. FATTY ACID SYNTHESIS IS REQUIRED FOR HER2+ BREAST CANCER BRAIN METASTASIS

Abstract Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2-positive breast cancer and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, resulting in site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase reduces human epidermal growth factor receptor 2-positive breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

BSCI-17. Targeting SIRPα as a therapeutic strategy for the treatment of breast cancer brain metastasis

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by the lack of druggable targets and an incidence of brain metastasis from the primary site of approximately 35%. There is no standard treatment for managing brain metastasis associated with TNBC; therefore, new strategies are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, targeting SIRPα as a potential strategy to mitigate tumor burden remains understudied. Analysis of gene expression database shows that SIRPα expression is significantly elevated in invasive breast cancer when compared to primary. Furthermore, single-cell data indicates that SIRPα is expressed in basal epithelial cells in TNBC tumors aside from the myeloid compartment. Our immune staining against SIRPα in patient biopsies shows a five-fold increase in SIRPα expression in metastatic brain tumors compared to the primary lesions. Therefore, targeting SIRPα may be a new immunotherapeutic strategy to treat breast cancer brain metastases. Anti-SIRPα treatment of mice bearing brain metastatic 4T1br3 orthotopic tumors showed reduced tumor volume and tumor weight by over 50% compared to isotype control-treated mice. Furthermore, in a model of intracardial brain metastasis, treatment with SIRPα antibody was associated with a 60% increase in survival compared to isotype control-treated mice. RNA sequencing of tumors indicated that SIRPα blockade is associated with a reduction in genes linked to mitochondrial respiratory chain and increases in negative regulation of the cell cycle. Furthermore, in vitro SIRPα targeting enhanced the cell-mediated cytotoxicity of microglia against 4T1Br3 breast cancer cells. This suggests that SIRPα blockade may influence both tumor and innate immune cells to limit brain metastatic breast cancer growth and enhance survival.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

The Target Differences of Anti-Tumorigenesis Potential of Curcumin and its Analogues Against HER-2 Positive and Triple-Negative Breast Cancer Cells.

Purpose: The current study aims to evaluate the in vitro cytotoxic and cell migration effects of synthetic curcumin and its analogues on HER2 and nuclear factor kappa B (NFκB) pathways, as well as the in vivo inhibitory effect on cancer growth of metastatic breast cancer. Methods: Cell viability, protein expression, and protein localization were determined in vitro using MTT assay, western blotting, and immunofluorescence, respectively. Meanwhile, scratch wound healing assay and gelatin zymography were conducted to investigate the metastasis inhibitory effect. The in vivo anti-tumor ability was evaluated in xenograft mouse model using triple-negative breast cancer (TNBC) cells. Results: Curcumin, PGV-0, and PGV-1 exhibited cytotoxic effect against HER2-overexpressing breast cancer cells. Although PGV-1 showed the best activity in the single cytotoxic assay, curcumin showed the strongest synergism with doxorubicin. Curcumin and PGV-0 inhibited membrane localization of HER2. In contrast, PGV-1 neither inhibited localization nor decreased the expression of HER2, nonetheless showed the most potent inhibition against nuclear localization of p65 indicating the different mechanisms of curcumin, PGV-0, and PGV-1. Regarding cancer metastasis, curcumin and PGV-1 showed inhibitory activities against cell migration and inhibited MMP-2 and MMP-9 protein expression. Lastly, PGV-1 was more potent compared to curcumin to suppress the tumor formation of metastatic breast cancer xenograft model in nude mice. Conclusion: Overall, our study strengthens the potency of curcumin analogue, PGV-1, for treating several types of cancer, including metastatic breast cancer.

Abstract 2603: Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling

Abstract Cancer inflammation promotes cancer progression, resulting in a high risk of cancer. Here, we demonstrate that our new herbal extract, SH003, suppresses both tumor growth and metastasis of MDA-MB-231 breast cancer cells via inhibiting STAT3-IL-6 signaling path. Our new herbal formula, SH003, mixed extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz, suppressed MDA-MB-231 tumor growth and lung metastasis in vivo and reduced the viability and metastatic abilities of MDA-MB-231 cells in vitro. Furthermore, SH003 inhibited STAT3 activation, which resulted in a reduction of IL-6 production. Therefore, we conclude that SH003 suppresses highly metastatic breast cancer growth and metastasis by inhibiting STAT3-IL-6 signaling path. Citation Format: Yu-Jeong Choi, Myeong-Sun Kim, SooYeon Kang, Seo Yeon Lee, Ji Hye Kim, Youn Kyung Choi, Sukjoong Oh, Seong-Gyu Ko. Herbal extract SH003 suppresses tumor growth and metastasis of MDA-MB-231 breast cancer cells by inhibiting STAT3-IL-6 signaling [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 2603.

SAT-337 Disruption Of The Circadian Melatonin Signal By Dim Light At Night Promotes Bone-lytic Breast Cancer Metastases

Breast cancer metastasis to bone is a major source of morbidity and mortality in women with advanced metastatic breast cancer. Morbidity from metastasis to bone is compounded by the fact that they cannot be surgically removed and can only be treated with chemotherapy and/or radiation therapy. Thus, there is critical need to develop new treatment strategies that kill bone metastatic tumors and reduce osteolytic lesions to improve patient quality of life and extend patient survival. Circadian rhythms are daily cycles of ~24 h that control many if not most physiologic processes and their disruption by exposure to light at night (LAN) or jet lag has been shown to be strongly associated with the development of cancer, particularly breast cancer. We have found that disruption of the anti-cancer circadian hormone melatonin (MLT) by light at night can significantly enhance the metastatic potential in breast cancer cells. Our work supports the report of the International Agency for Research on Cancer that shift work is a “probable human carcinogen” and highlights the association between exposure to light at night and invasive breast cancer. We recently reported that human breast tumor xenografts grown in athymic nude female rats housed in a photoperiod of 12h light at day: 12h dim light at night (dLAN, 0.2 lux - blocks the nighttime circadian MLT signal), display resistance to doxorubicin (Dox). More importantly, tumor growth and drug resistance could be blocked by the administration of Dox in circadian alignment with nocturnal MLT during dLAN. Our recent preliminary studies show that poorly invasive ERα positive MCF-7 breast cancer cells, when injected into the tibia (to mimic bone metastatic disease) of Foxn1nu athymic nude mice (which produce a strong circadian nighttime melatonin signal) housed in a dLAN photoperiod (suppressed nocturnal MLT production) developed full blown breast cancer tumors in bone (P<0.05) that are highly osteolytic (P<0.05). Moreover, patients with metastatic breast cancer are routinely treated with doxorubicin, which itself can promote bone damage. Our studies demonstrate that MLT slows the growth of metastatic breast cancer in bone but that the chrono-therapeutic use of doxorubicin in circadian alignment with melatonin in Foxn1nu mice with tibial breast tumors, reduced tumor growth in bone, reduced bone erosion, and promoted the formation of new bone. Successful use of this chronotherapeutic use of Dox and MLT in clinical trials increasing efficacy in preventing or suppressing breast cancer metastasis to bone while decreasing toxic side effects of doxorubicin would provide a revolutionary advancement in the treatment of bone metastatic breast cancer and decrease the morbidity and mortality associated with breast cancer metastasis to bone. Acknowledgements: Louisiana Clinical and Translational Science Center (LACATS) visiting scholar program award and pilot grant to MA.

Inhibition of the Stromal p38MAPK/MK2 Pathway Limits Breast Cancer Metastases and Chemotherapy-Induced Bone Loss.

The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss.Significance: Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. Cancer Res; 78(19); 5618-30. ©2018 AACR.

Abstract P3-06-02: Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors

Abstract Background. Despite medical advances, currently there is no treatment for breast to bone metastasis. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Pharmacokinetic and pharmacodynamic studies were performed. Plasma and bone marrow supernatants were collected from PyMT-R221A tumor bearing mice treated with ML115 (5mg/Kg) at 0.25, 0.5, 1, 2, 4, 8, 24 hours and three weeks (n=3 mice/time point). Currently, we are investigating the BMMPIs ability to impact the metastatic process through an in vivo model of intracardiac inoculation. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p&amp;lt;0.05) compared to control. In vivo, BMMPIs significantly reduced the growth of bone metastatic breast cancer compared to control and the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p&amp;lt;0.05). ML115 is rapidly cleared from the plasma and accumulates selectively in the bone marrow microenvironment over time. Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Soliman HH, Lynch CC. Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-02.

Abstract 4901: Urokinase-derived peptide UP-7 effectively inhibits metastatic growth of breast cancer through suppression of FAK activation

Abstract The recombinant kringle domain of urokinase-type plasminogen activator (UK1) has been shown to inhibit angiogenesis in vitro and in vivo and suppress brain tumor in vivo. To avoid limitations in application due to mass production of recombinant proteins, we dissected UK1 sequences to seven peptides based on structure and amino acid characteristics, and examined the anti-angiogenic activities for the constructed peptides. UP-7 peptide derived from ß-sheets region of UK1 was the most potent inhibitor of the proliferation and migration of endothelial cells (ECs) in vitro, and it also inhibited in vivo angiogenesis in the mouse matrigel plug assay. Such anti-angiogenic activities were not exerted by the scrambled peptide. At molecular level, UP-7 inhibited the VEGF or bFGF-induced phosphorylation of FAK and ERK1/2 and suppressed formation of stress fibers and focal adhesions. It also inhibited the attachment and spreading of ECs onto immobilized fibronectin. In a lung cancer animal model xenografted with UP-7-non-sensitive NCI H460, systemic treatment of UP-7 effectively suppressed tumor growth through inhibition of angiogenesis. Interestingly, breast cancer cells such as MDA-MB231 were sensitive to UP-7 in proliferation differently from other cancer cells. It inhibited the migration and invasion of LM-2-MDA-MD231 and suppressed the phosphorylation of FAK. Accordingly, UP-7 potently inhibited lung metastatic growth of LM2-MDA-MB-231 in experimental metastasis model. Taken together, these results suggest that novel UK1-derived peptide, UP-7 can be effectively used for treatment of the metastatic growth of breast cancer. (NRF-2012R1A1A2007175, NRF-2012R1A5A2051476) Citation Format: Young Ae Joe, Hyun-Kyung Kim, Purevjargal Naidansuren, Seung Woo Lee, Rae-Kwon Kim, Su-Jae Lee, Suk Keun Lee, Yong-Kil Hong. Urokinase-derived peptide UP-7 effectively inhibits metastatic growth of breast cancer through suppression of FAK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4901. doi:10.1158/1538-7445.AM2017-4901

Abstract 2963: Inhibition of stromal p38MAPK abrogates breast cancer metastasis

Abstract Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that its inhibition significantly reduced breast cancer bone metastases by specifically targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone. Citation Format: Sheila A. Stewart, Bhavna Murali, Qiaho Ren, Xinming Su, Kevin Flannagan, Xianmin Luo, Jasmine Sponagel, Yujie Fu, Elise Alspach, Kathlees Leahy, Roberta Faccio, Kathleen Weilbaecher, Joseph Monahan. Inhibition of stromal p38MAPK abrogates breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2017-2963

Abstract P6-12-10: Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth

Abstract Bone metastasis is a common event during breast cancer progression. The resultant lesions are painful and currently, despite medical advances, are incurable. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale: Gene expression analysis and validation in human and murine specimens of bone metastases revealed that matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment and significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor or host derived MMP-2 contributes to breast cancer growth and does so by processing substrates including type I collagen and transforming growth factor beta (TGFβ) latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. Methods: We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). Results: In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells, (PyMT, 4T1, MDA-MB-231, MCF-7), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, we demonstrated using two bone metastatic models (PyMT-R221A-Luc and 4T1-Luc) that BMMPI treatment significantly reduced tumor growth and tumor associated bone destruction. Additionally, BMMPIs are superior in promoting tumor apoptosis compared to the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p&amp;lt;0.05). We demonstrated MMP-2 selective inhibition in the bone microenvironment using specific and broad spectrum MMP probes. Further, compared to zoledronate, BMMPI treated mice had significantly lower levels of TGFβ signaling and MMP generated type I collagen carboxy-terminal (ICTP) fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. Conclusions. MMP-2 specific inhibition was achieved in the bone microenvironment. BMMPIs significantly inhibit breast cancer growth in bone, they are able to induce breast cancer cell apoptosis and prevent cancer induced bone destruction. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Lynch CC. Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-10.

Abstract 3374: GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity

Abstract We recently described a human cell population with progenitor-like phenotype (CD45-CD34+), resident in the white adipose tissue (WAT) and able to promote local and metastatic breast cancer (BC) progression and angiogenesis (Orecchioni et al., 2013). The molecular mechanism involved in this interaction has been so far elusive. An extensive screening of candidate molecules related to angiogenesis, inflammation, motility and invasiveness revealed that two proteins are significantly up-regulated in WAT-derived progenitors following culture with BC cells: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Matrix metallopeptidase 9 (MMP9). In vivo, both proteins were overexpressed in orthotopic models of human BC co-injected with human WAT progenitors. The inhibition of GM-CSF by a monoclonal antibody in diet-induced, obese BC mice led to a reduced intratumor vascularization and a strong impairment of WAT immunosuppressive microenvironment, targeting mainly myeloid cells such as macrophages and myeloid derived suppressor cells (MDSCs) in peritumoral WAT. Circulating levels of monocytes and of CD4+CD25brightCD127low/neg T-regulatory cells (T-regs) were also reduced in treated mice. Similarly, soluble immunosuppressive factors, such as IL-10 and IL-5, and CD274 (PD-L1) were reduced in tumors and WAT collected from immune competent mice neutralized for GM-CSF, confirming the crucial role of the factor in promoting tumor immune escape. This resulted in a significantly reduced local BC growth and lower metastatic progression in vivo. All these findings challenge the clinical use of GM-CSF. In the same syngeneic model, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth, without altering immune cells composition in tumor microenvironment. The combined inhibition of GM-CSF and MMP9 was synergic in impairing angiogenesis, local and metastatic BC growth in diet-induced obese orthotopic BC models, indicating a potential complementary role in tumor spread. As we recently reported that Metformin targets both BC cells and the neoplastic WAT environment (Orecchioni et al., 2015), we investigated Metformin effect over GM-CSF and MMP9 expression. Metformin inhibited GM-CSF and MMP9 up-release from WAT progenitors in vitro. Circulating GM-CSF was significantly impaired in BC xenografts administered with Metformin and MMP9 expression was also affected by the treatment. Collectively these results indicate GM-CSF and MMP9 as key candidates involved in the pro-tumorigenic effect of WAT progenitors on BC in a setting of obesity. The comparison between Metformin and GM-CSF/MMP9 specific inhibition is currently under investigation. Citation Format: Francesca Reggiani, Valentina Labanca, Giovanna Talarico, Stefania Orecchioni, Patrizia Mancuso, Francesco Bertolini. GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity. [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 3374.

Abstract LB-270: Inhibition of the stromal p38MAPK pathway abrogates breast cancer metastases

Abstract Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that this inhibition significantly reduced breast cancer bone metastases by targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition specifically targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our clinically relevant preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone. Citation Format: Sheila A. Stewart, Bhavna Murali, Xinming Su, Kevin Flanagan, Jasmin Sponagel, Xianmin Luo, Yujie Fu, Elise Alspach, Kathleen Leahy, Joseph Monahan, Katherine Weilbeacher. Inhibition of the stromal p38MAPK pathway abrogates breast cancer metastases. [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 LB-270.

Abstract P6-16-02: Treatment of skeletal metastatic breast cancer with bone seeking matrix metalloproteinase inhibitors

Abstract Background. Breast to bone metastasis is a common event during breast cancer progression. The resultant lesions are painful and currently, despite medical advances, are incurable. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. Given that previous broad-spectrum MMP inhibitor (MMPI) trials were unsuccessful due to dose limiting systemic side effects, we utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p&amp;lt;0.05) compared to control. In vivo BMMPIs significantly reduced the growth of bone metastatic breast cancer compared to control and the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p&amp;lt;0.05). Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Lynch CC. Treatment of skeletal metastatic breast cancer with bone seeking matrix metalloproteinase inhibitors. [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 P6-16-02.

Activin-A signaling promotes epithelial-mesenchymal transition, invasion, and metastatic growth of breast cancer.

Background:Activins belong to the transforming growth factor-β (TGF-β) superfamily of cytokines. Although the role of TGF-β in cancer progression has been highly advocated, the role of activin signaling in cancer is not well known. However, overexpression of activin-A has been observed in several cancers.Aims:The gene expression profile indicated higher expression of Activin-A in breast tumors. Hence the aim of this study was to evaluate the status and role of Activin signaling pathway in these tumors.Methods:Microarray analysis was performed to reveal gene expression changes in breast tumors. The results were validated by quantitative PCR and immunohistochemical analysis in two independent sets of normal and tumor samples. Further, correlation of activin expression with survival and distant metastasis was performed to evaluate its possible role in tumor progression. We used recombinant activin-A, inhibitors, overexpression, and knockdown strategies both in vitro and in vivo, to understand the mechanism underlying the protumorigenic role of this signaling pathway.Results:We report that activin-A signaling is hyperactivated in breast cancers as indicated by higher activin-A, phosphoSMAD2, and phosphoSMAD3 levels in advanced breast cancers. Bone morphogenetic proteins and molecules involved in this signaling pathway were downregulated, suggesting its suppression in breast cancers. Activin-A expression correlates inversely with survival and metastasis in advanced breast cancers. Further, activin-A promotes anchorage-independent growth, epithelial–mesenchymal transition, invasion, angiogenesis, and stemness of breast cancer cells. We show that activin-A-induced phenotype is mediated by SMAD signaling pathway. In addition, activin-A expression affects the tumor-forming ability and metastatic colonization of cancer cells in nude mice.Conclusions:These results suggest that activin-A has a critical role in breast cancer progression and, hence, targeting this pathway can be a valuable strategy in treating breast cancer patients.

Abstract 5214: Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells

Abstract We have recently described that the human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression (Martin-Padura et al, 2012; Orecchioni et al, 2013). The biguanide metformin (met), commonly used for type 2 diabetes, might have activity against BC and we found it able to inhibit angiogenesis in vivo (Dallaglio et al, 2014; Orecchioni et al, 2014). We studied met and another biguanide, phenformin (phe), in vitro and in vivo in orthotopic NSG murine models of local and metastatic BC. As met is frequently administered with aspirin or atenolol in diabetic/obese patients, we studied in vitro and vivo their association. In vitro, biguanides activated AMPK, inhibited complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. Aspirin was synergistic with met and phe in inducing apoptosis of estrogen receptor+ BC cells. This synergistic effect was less evident in triple negative BC cells. In co-culture, biguanides significantly inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides also inhibited local and metastatic BC growth in a genetically engineered model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and impaired of the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. This effect was significantly increased by the addition of aspirin or atenolol. This was evident also when AMPK activation was assessed in combination therapy regimens. AMPK phosphorylation was significantly increased in BC cells treated with met+aspirin or phe+atenolol as compared to met or phe alone. In WAT progenitors, AMPK activation was enhanced only under met+aspirin and met+atenolol combinations. In immune-competent mice, met effect in BC models was significantly enhanced by the addition of atenolol or of aspirin. Phe was significantly more active than met both in vitro and in vivo. Considering their safety profile, biguanides (alone or in combination with aspirin or atenolol) deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. Citation Format: Giovanna Talarico, Francesca Reggiani, Stefania Orecchioni, Patrizia Mancuso, Angelica Calleri, Giuliana Gregato, Valentina Labanca, Douglas M. Noonan, Katiuscia Dallaglio, Adriana Albini, Francesco Bertolini. Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. [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 5214. doi:10.1158/1538-7445.AM2015-5214

Abstract 4231: Combination of epigenetic, differentiation and DNA damaging agents induce tumor cell death and stem cell depletion in breast cancer

Abstract The histone deacetylase inhibitor, entinostat, is a new-generation epigenetic drug, which has recently demonstrated notable clinical efficacy when used in combination with standard therapy. Retinoids induce differentiation in various types of stem cells. However, its delivery to patients is challenging because of its rapid metabolism. Also, epigenetic changes in the retinoic acid receptors often render cancer cells retinoid-resistant. We have shown that a combination of entinostat, all-trans retinoic acid (ATRA) and doxorubicin causes significant regression of xenografts of triple negative breast cancer (TNBC) cells and investigated the mechanism underlying the effectiveness of this combination therapy. Combinations of entinostat, retinoic acid and doxorubicin were optimal in causing tumor regression in triple negative breast cancer xenografts. Gene expression analysis of treated TNBC cells identified genes most effectively reprogrammed by entinostat and doxorubicin (ED) combination therapy. These genes are involved in cell cycle arrest, inflammation and differentiation, which are related to better survival outcome in patients. Entinostat sensitizes the cells to doxorubicin-induced growth arrest, resulting in increased apoptosis and necrosis. Adding ATRA to ED regulated interferon genes and members of the cancer/testis antigens (CTA) and tripartite motif (TRIM) family of proteins and induces inflammation in nude mice. Entinostat/ATRA/dox therapy was most effective to target breast cancer stem cells (BCSC) in limiting dilution assays of growth in mammary fat pads. The epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate cellular proliferation and differentiation mediates the epigenetic differentiation effect. Patient-derived distant metastases responded to entinostat/ATRA/dox therapy in culture. Thus, the combination therapy may have significant effects in decreasing both local and metastatic growth in breast cancer, especially in decreasing recurrence by targeting breast cancer stem cells. Citation Format: Vanessa F. Merino, Nguyen Nguyen, Helen Sadik, Soonweng Cho, Leslie Cope, Xian C. Zhou, Zhe Zhang, Qian Chen, Duojia Pan, David L. Huso, Syed Ali, Christina Adams, Balázs Győrffy, Saraswati Sukumar. Combination of epigenetic, differentiation and DNA damaging agents induce tumor cell death and stem cell depletion in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2015-4231