1084 Background: Brain metastasis is the most common malignancy of the central nervous system which causes severe morbidity and mortality in multiple cancer types of patients and represents an unmet medical need. Several critical steps are required for a successful brain metastasis, including local invasion, intravasation, dissemination, extravasation, and colonization. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information toward how cancer cells extravasate and colonize. Methods: To understand the underlying molecular mechanisms and applications of molecular targets for brain metastasis therapy, murine models are employed for investigation of brain extravasation and colonization. To investigate the roles of SQLE in breast-to-brain metastasis in vivo, we silenced SQLE expression directly with lentiviral shRNA in the brain metastatic MDA-MB-231-BR cell line (231-BR/shSQLE) and used 231-BR cells expressing a scramble shRNA (231-BR/shScr) as the control. Brain metastases were induced by intracardiac, orthotopic, or direct intracranial injections of 231-BR/shSQLE or 231-BR/shScr cells into immune deficient mice. The essential roles of SQLE in the specific step(s) of breast-to-brain metastatic process were evaluated by ex vivo immunofluorescence analysis of brain slices from the animals. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast-to-brain metastasis, we evaluated the inhibitory effects of NB-598 (a SQLE inhibitor) in both the 231-BR orthotopic and intracardiac mouse models. Results: Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in the cholesterol biosynthesis, plays a critical role in the processes of breast cancer metastasized to the brain, especially in brain extravasation and colonization. Interestingly, the pharmacologic inhibition of SQLE has been widely used against fungal infections, and the next-generation SQLE inhibitors have been recently shown to exert an anticancer effect. Our data demonstrated that SQLE is essential for 231-BR cells to extravasate into the parenchyma as well as the formation of micro and macro-metastases in the brain. Interestingly, we found that astrocytes play a key role in supporting 231-BR-developed brain macro-metastasis. In vitro blood-brain barrier (BBB) models further demonstrated the critical roles of SQLE in promoting 231-BR cell invasion and penetration through BBB. Inhibition of SQLE by NB-598 demonstrated anti-tumor proliferation and anti-metastasis. Conclusions: Pharmacologic inhibition of SQLE by NB-598 suppressed 231-BR tumor growth at the mammary fat pad and distal metastases to organs, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer metastases.
e13101 Background: Brain metastases are serious complications of breast cancer and there is no effective treatment. The major therapeutic issues can be attributed to the unique brain microenvironment as well as the difficulty of drug delivery to the brain due to the blood-brain barrier (BBB). BBB limits access of nutrients from the circulation and thereby makes the brain in a special condition that is hypoxic and composed of depleted metabolites, growth factors, and proteins. Nevertheless, tumor cells that are able to metastasize to the brain can necessitate metabolic adaptations for their nutrient availability and thereby succeed to colonize in the brain. However, it is still poorly understood how breast cancer cells alter their metabolic systems to manipulate brain metastasis by overcoming nutritional limitations. Methods: To probe the molecular mechanism of breast cancer to brain metastasis, we compared gene signatures in primary breast tumors and breast-to-brain metastatic tumors, and identified that SQLE (encodes squalene epoxidase, the second rate-limiting enzyme in the cholesterol biosynthesis) could be a leading-edge gene in breast-to-brain metastasis. Using the brain metastatic MDA-MB-231-BR (231-BR) and its parental MDA-MB-231 (231) cell line expressing SQLE shRNAs, we evaluated the effects of SQLE loss on cancer cell migration, invasion, and stemness by wound-healing, transwell invasion/migration, and tumorsphere formation assays. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain metastases were established to verify our hypothesis. Results: While loss of SQLE greatly attenuated cell invasiveness and stemness in both 231 and 231-BR cells, loss of SQLE could only affect the cell migration activity on 231 cells but not 231-BR cells. Since the ability to invade, migrate, and penetrate is critical for invasion of cancer cells, our results strongly imply the novel function of SQLE in breast cancer cell invasion, penetration, and colonization in the brain. Our RNA-seq data further identified a subset of SQLE-affected genes that is uniquely enriched in 231-BR cells and favors brain extravasation and colonization. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain extravasation and colonization finally verified our hypothesis that SQLE may play a unique function to promote breast cancer metastasis into the brain. Conclusions: Our findings provide new insights into contributions by SQLE in breast-to-brain extravasation and colonization and indicate that targeting SQLE may represent a therapeutic opportunity for breast cancer brain metastases.
Tamoxifen is a widely known estrogen receptor (ER) modulator which has been employed in adjuvant treatment of ER+ breast cancer for over 30 years. Interestingly, clinical observations reveal that tamoxifen is capable of inducing regression of some tumors lacking ER expression whereas tamoxifen is also capable of increasing host resistance against cancer in an ER-independent mechanism. These findings suggest the immunomodulatory effects of tamoxifen may be ER-independent, but little is known about the underlying mechanism and the potential clinical implication. Recently, we identified a novel mechanism by which tamoxifen exerts its DNA-damaging potential by re-shaping the unfavorable tumor microenvironment in breast cancer. A long-term tamoxifen administration induces downregulation of the chromatin ‘‘reader’’ RACK7/ZMYND8, which acts as a suppressor of interferon-stimulated genes (ISGs, including cytokines and chemokines) and CEACAM1 in both ER+ and triple negative breast cancer (TNBC) cells. To investigate the immunomodulatory effects of tamoxifen in conjunction with RACK7-knockdown, the orthotopic murine TNBC 4T1 model was employed to investigate tamoxifen-mediated cellular modulation in TNBC. The control and RACK7-knockdown 4T1 cells are orthotopically implanted into the mammary fat pad of female BALB/c mice. Peripheral cytokines/chemokines and high-content biomarker studies (multiplex immunoassays, flow cytometry, and single-cell RNA sequencing) are deployed to obtain insights into the mechanistic rationale behind the immunomodulatory effects of tamoxifen and/or RACK7-knockdown. The tamoxifen-mediated cellular modulation evokes cytokine/chemokine secretion and further induces T-cell infiltration into tumor area. However, tumor reduction was limited due to extensive T-cell exhaustion from interaction of CEACAM1 and TIM-3, a “checkpoint” receptor expressed in CD4+ and CD8+ T cells. The expression patterns of CEACAM1 and PD-L1 in 4T1 tumor cells and that of TIM-3 and PD-1 in CD4+ and CD8+ T-cells correlate with intra-tumor infiltration of T-cells and tumor cell growth. Therefore, targeting the interaction between CEACAM1 and TIM-3 to overcome T-cell exhaustion is crucial for the new therapeutic role of tamoxifen treatment in TNBC breast cancer in conjugation with RACK7-knockdown. Altogether, our findings provide direct evidence to support a new therapeutic opportunity by targeting CEACAM1-TIM-3 interaction in the tamoxifen-mediated tumor immune microenvironment for improving immune checkpoint blockade therapy in breast cancer. Citation Format: Pony Yu-Ling Lee, Marvin A. Aberin, Chien-Chang Shen, Kun-Yuan Lin, Chao Di Chang, Chih-Chieh Yang, Shan-Yun Cheng, Ya Wen Hung, Xin-Guo Hsu, Shu-Ping Wang. Reshaping the tumor microenvironment: new application of tamoxifen in triple negative breast cancer immunomodulation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5953.
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