Triple-negative Breast Cancer Lung Metastasis Research Articles

Abstract P2-15-03: Liposomal annamycin inhibition of lung localized breast cancer

Abstract Background and Rationale: Annamycin is an anthracycline antibiotic currently in clinical studies in AML patients. Our earlier studies showed that Annamycin is not cross-resistant with doxorubicin (DOX) and is a poor substrate for P-glycoprotein 1 (P-gp) [aka ATP-binding cassette sub-family B member 1 (ABCB1) or multidrug resistance protein 1 (MDR1)], a major mechanism of doxorubicin resistance in different types of cancer. Annamycin, in contrast to DOX, achieves relatively high levels of cellular accumulation, especially in multidrug resistant (MDR) cell lines, and induces significant DNA damage in MDR cancer cells. Clinically, Annamycin is administered in a liposomal formulation (L-Annamycin) the in vivo activity of which has been demonstrated in different tumor models. IND aimed preclinical studies revealed that in sharp contrast to DOX, Annamycin displays dramatically reduced to no cardiotoxicity. Subsequent pharmacokinetic and organ distribution studies revealed surprisingly high levels of Annamycin in lung tissue. Annamycin levels (AUC, 24 h) in lungs were over 10-fold greater than in plasma. In addition, when compared with DOX, lung levels of Annamycin were 6-fold higher than those achievable with DOX. Thus, studies aimed to assess efficacy of L-Annamycin in lung localized tumor models were initiated. Objective: The objective of this study was to assess the in vitro and in vivo activity of L-Annamycin in 4T1, a highly aggressive triple negative breast cancer (TNBC) lung colonization model. Methods: In vitro efficacy of Annamycin was investigated in a panel of TNBC cell lines including murine 4T1 and human MDA-MB 231, MDA-MB-468, BT20, and SUM149 cell lines. Uptake and subcellular distribution of Annamycin was assessed using fluorescent microscopy and FACS. Biodistribution of Annamycin was tested in naïve CD-1 mice after intravenous administration of the drug. Efficacy of L-Annamycin was tested in a syngeneic TNBC lung model, specifically, in immune-competent Balb/c mice injected intravenously with 4T1 cells expressing firefly luciferase. Results: Annamycin shows consistently high cytotoxicity across all tested cell lines with IC50s in the low nanomolar range. Uptake analysis performed on 4T1 cells indicated significantly higher uptake of the drug as compared to DOX. Annamycin shows distinct subcellular distribution with high cytosolic deposition, while DOX was mostly confined to the nucleus. In vivo efficacy studies in mice treated with L-Annamycin showed significantly increased survival when compared to vehicle treated animals. Delayed tumor progression was assessed by bioluminescent imaging and correlated with increased survival. Conclusion: Our in vitro studies demonstrate the high potency of L-Annamycin in vitro and in vivo. Its unique intracellular uptake and distribution might in part be responsible for L-Annamycin’s activity profile and lack of cardiotoxicity. The observed high in vivo efficacy of L-Annamycin in an aggressive TNBC lung metastasis model coupled with consistently high accumulation of L-Annamycin in lung parenchyma, may prove to open new therapeutic options for patients with tumors metastatic to the lung. Citation Format: Rafal Zielinski, Krzysztol Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Adam Didluch, Waldemar Priebe. Liposomal annamycin inhibition of lung localized breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-15-03.

Abstract P3-02-15: Integrin a2 promotes stemness and lung metastasis in triple negative breast cancer

Abstract Accumulating evidence demonstrates that cancer plasticity and stem cell properties (stemness) drive both tumorigenesis and metastasis, and how to eliminate metastasis remains one of the biggest challenges in the clinic. Here we report that the integrin receptor CD49b-encoding ITGA2 is an oncogenic target of miR-206 regulating cancer stemness and metastasis through pathways linked to lipid metabolism and cell cycle regulation in metastatic triple negative breast cancer (TNBC). ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of TNBC. RNA sequencing analyses revealed that ITGA2 knockdown inhibits newly identified pathways such as lipid metabolism and protein kinase activity. ACLY-encoded ATP citrate lyase is one of the top targets in CD49b promoting lipid biosynthesis and self-renewal of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. High expression levels of the ITGA2/ACLY/CCND1 axis are associated with an unfavorable relapse-free survival of patients with high grade breast cancer, in both basal-like and other subtypes. This study identifies ITGA2 as a potential therapeutic target of TNBC stemness, metabolism, and metastasis. Citation Format: Valery Adorno-Cruz, Andrew Hoffmann, Xia Liu, Ruth Keri, Huiping Liu. Integrin a2 promotes stemness and lung metastasis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-15.

Integration of whole-genome sequencing and functional screening identifies a prognostic signature for lung metastasis in triple-negative breast cancer.

Lung metastasis is one of the leading causes of death for triple-negative breast cancer (TNBC). We sought to characterize the genetic alterations underlying TNBC lung metastases by integrating whole-genome sequencing and functional screening. Furthermore, we aimed to develop a metastasis-related gene signature for TNBC patients to improve risk stratification. In this prospective observational study, we first conducted whole-genome sequencing of paired primary tumor and lung metastasis from one TNBC patient to identify potential genetic driver alterations. An in vivo gain-of-function screening using an amplified open reading frame library was then employed to screen candidate genes promoting lung metastasis. Finally, we applied Cox proportional hazard regression modeling to develop a prognostic gene signature from 14 candidate genes in TNBC. Compared to the primary tumor, copy number amplifications of chromosomes 3q and 8q were identified in the lung metastasis. We discovered an enrichment of 14 genes from chromosomes 3q and 8q in mouse lung metastases model. We further developed and validated a four-gene signature (ENY2, KCNK9, TNFRSF11B and KCNMB2) that predicts recurrence-free survival and lung metastasis in TNBC. Our data also demonstrated that upregulated expression of ENY2 could promote invasion and lung metastasis of TNBC cells both in vitro and in vivo. In conclusion, our study reveals functional genes with copy number amplifications among chromosome 3q and 8q in lung metastasis of TNBC. And we develop a functional gene signature that can effectively stratify patients into low- and high-risk subgroups of recurrence, helping frame personalized treatments for TNBC.

Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus, it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC. To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor. E6201 inhibited TNBC cell colony formation, migration, and invasion in a dose-dependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201's in vitro anti-tumor efficacy. These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and anti-metastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.

P211 - Integration of whole-genome sequencing and functional screening identifies a prognostic signature for lung metastasis in triple-negative breast cancer

Lung metastasis is one of the leading causes of death for triple-negative breast cancer (TNBC). We sought to characterize the genetic alterations underlying TNBC lung metastases by integrating whole-genome sequencing and functional screening. Furthermore, we aimed to develop a metastasis-related gene signature for TNBC patients to improve risk stratification. In this prospective observational study, we first conducted whole-genome sequencing of paired primary tumor and lung metastasis from one TNBC patient to identify potential genetic driver alterations. An in vivo gain-of-function screening using an amplified open reading frame library was then employed to screen candidate genes promoting lung metastasis. Finally, we applied Cox proportional hazard regression modeling to develop a prognostic gene signature from 14 candidate genes in TNBC. Compared to the primary tumor, copy number amplifications of chromosomes 3q and 8q were identified in the lung metastasis. We discovered an enrichment of 14 genes from chromosomes 3q and 8q in mouse lung metastases model. We further developed and validated a four-gene signature (ENY2, KCNK9, TNFRSF11B and KCNMB2) that predicts recurrence-free survival and lung metastasis in TNBC. Our data also demonstrated that upregulated expression of ENY2 could promote invasion and lung metastasis of TNBC cells both in vitro and in vivo. In conclusion, our study reveals functional genes with copy number amplifications among chromosome 3q and 8q in lung metastasis of TNBC. And we develop a functional gene signature that can effectively stratify patients into low- and high-risk subgroups of recurrence, helping frame personalized treatments for TNBC.

Activated Platelets‐Targeting Micelles with Controlled Drug Release for Effective Treatment of Primary and Metastatic Triple Negative Breast Cancer

AbstractThe frequent relapse and metastasis characteristics of triple negative breast cancer (TNBC) make it a fraught issue with very poor prognosis in clinic. An effective treatment for TNBC should prevent and even eliminate metastasis as well as suppress primary lesion expansion. Recent progress reveals that platelets can be recruited and activated by tumor cells through intercellular adhesion molecules (ICAM), and help aggressive circulating tumor cells (CTCs) form metastasis. Therefore, activated platelets are considered with possession of tumor‐homing, CTC‐capturing, and metastasis‐targeting abilities. In this work, a P‐selectin (expressed on activated platelet surface) targeting peptide (PSN) is modified on a redox‐responsive paclitaxel‐loaded micelle (PSN‐PEG‐SS‐PTX4 micelle) to utilize activated platelets as a “bridge” for interaction with cancer cells. The PSN‐modified micelle can easily adhere to the surface of activated platelets and subsequently capture CTCs in blood circulation. Compared to Taxol and PEG‐SS‐PTX4 micelle, PSN‐PEG‐SS‐PTX4 micelle also exhibits enhanced primary TNBC/metastasis targeting and penetrating effect through binding with tumor infiltrating platelets and thus significantly improves treatment outcome. More importantly, PSN‐PEG‐SS‐PTX4 micelle potently suppressed lung metastasis of TNBC and reduced incidence of distant liver metastasis. The activated platelet‐targeting redox‐responsive micelle system provides a promising prospect for the omnidirectional treatment of metastatic cancer.

Repurposing suramin for the treatment of breast cancer lung metastasis with glycol chitosan-based nanoparticles

Suramin (SM), a drug for African sleeping sickness and river blindness therapy, has been investigated in various clinical trials for cancer therapy. However, SM was eventually withdrawn from the market because of its narrow therapeutic window and the side effects associated with multiple targets. In this work, we developed a simple but effective system based on a nontoxic dose of SM combined with a chemotherapeutic agent for the treatment of metastatic triple-negative breast cancer (TNBC). SM and glycol chitosan (GCS) formed nanogels because of the electrostatic effect, whereas doxorubicin (DOX) was incorporated into the system through the hydrophilic and hydrophobic interactions between DOX and GCS as well as the ionic interactions between DOX and SM to yield GCS-SM/DOX nanoparticles (NPs). GCS-SM/DOX NPs have a size of approximately 186 nm and a spherical morphology. In vitro experiments showed that GCS-SM NPs could effectively inhibit cancer cell migration and invasion, as well as angiogenesis. Furthermore, in a TNBC lung metastasis animal model, GCS-SM/DOX NPs significantly reduced tumor burden and extended the lifespan of animals, while not inducing cardio and renal toxicities associated with the DOX and SM, respectively. As all the components used in this system are biocompatible and easy for large-scale fabrication, the GCS-SM/DOX system is highly translatable for the metastatic breast cancer treatment. Statement of SignificanceThe doxorubicin-loaded glycol chitosan-suramin nanoparticle (GCS-SM/DOX) is novel in the following aspects: SM acts as not only a gelator for the first time in the preparation of the nanoparticle but also an active pharmaceutical agent in the dosage form. GCS-SM/DOX NP significantly reduced tumor burden and extended the lifespan of animals with triple-negative breast cancer lung metastasis. GCS-SM/DOX NPs attenuate cardio and renal toxicities associated with the DOX and SM. The GCS-SM/DOX system is highly translatable because of its simple, one-pot, and easy-to-scale-up preparation protocol.

Anti-tumor responses to hypofractionated radiation in mice grafted with triple negative breast cancer is associated with decorin induction in peritumoral muscles.

Triple negative breast cancer (TNBC) is the most lethal one for all types of breast cancer. Though radiotherapy is an efficient treatment, long-term survival rate of TNBC patients is still suboptimal. Hyprofractionated radiotherapy, an improved radiotherapy, has made an inspiring result in clinic. However, the mechanism underlying TNBC treated with hyprofractionated radiotherapy is not clear. Decorin (DCN) is a small poteoglycan of matrix which has an inhibitory effect on the breast cancer and is secreted by muscle under certain conditions. In this study, we demonstrated that peritumoral muscles secrete more DCN at higher dose irradiation than that at conventional irradiation dose in TNBC tumor-bearing mice. Thus, it indicates that DCN secreted from peritumoral muscle may be one of the reasons why hyprofractionated radiotherapy could inhibit the growth of TNBC more effectively. Moreover, we also indicated that the up-regulated DCN attenuated lung metastasis of TNBC. In conclusion, we demonstrated that hypofractionated radiation promotes the secretion of DCN in peritumoral muscle, thus enhancing the inhibitory effect on TNBC, which might help to optimize the strategy of radiotherapy for TNBC patients in the future.

TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer that has limited therapeutic options. Thus, developing novel treatments for metastatic TNBC is an urgent need. Here, we show that nanoparticle-mediated delivery of transforming growth factor-β1-activated kinase-1 (TAK1) inhibitor 5Z-7-Oxozeaenol can inhibit TNBC lung metastasis in most animals tested. P38 is a central signal downstream of TAK1 in TNBC cells in TAK1-mediated response to multiple cytokines. Following co-culturing with macrophages or fibroblasts, TNBC cells express interleukin-1 (IL1) or tumor necrosis factor-α (TNFα), respectively. Compared to TAK1 inhibition, suppressing IL1 signaling with recombinant IL1 receptor antagonist (IL1RA) is less efficient in reducing lung metastasis, possibly due to the additional TAK1 signals coming from distinct stromal cells. Together, these observations suggest that TAK1 may play a central role in promoting TNBC cell adaptation to the lung microenvironment by facilitating positive feedback signaling mediated by P38. Approaches targeting the key TAK1-P38 signal could offer a novel means for suppressing TNBC lung metastasis.

A novel long non-coding RNA linc-ZNF469-3 promotes lung metastasis through miR-574-5p-ZEB1 axis in triple negative breast cancer.

Triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. The major sites for TNBC metastasis include the lungs, brain, liver, and bone. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides and have been reported as important regulators in BC metastasis. However, the underlying mechanisms for lncRNAs regulating TNBC metastasis are not fully understood. Here we found that linc-ZNF469-3 was highly expressed in lung-metastatic LM2-4175 TNBC cells and overexpression of linc-ZNF469-3 enhanced invasion ability and stemness properties in vitro and lung metastasis in vivo. Furthermore, we found linc-ZNF469-3 physically interacted with miR-574-5p and overexpression of miR-574-5p attenuated ZEB1 expression. Importantly, endogenous high expressions of linc-ZNF469-3 and ZEB1 were correlated with tumor recurrence in TNBC patients with lung metastasis. Taken together, our findings suggested that linc-ZNF469-3 promotes lung metastasis of TNBC through miR-574-5p-ZEB1 signaling axis and may be used as potential prognostic marker for TNBC patients.

A novel Ubc9 -dependent pathway regulates SIRT1- ER-α Axis and BRCA1-associated TNBC lung metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease and has a higher rate of recurrence and distant metastasis. African-American (AA) women have a higher frequency of BRCA1 mutations and TNBC compared to other populations. Basal-like tumors have a higher rate of brain, lung and distant nodal metastasis more than other TNBC subtypes, contributing to higher mortality rate. Our previous work suggested Ubc9, a SUMO E2-conjugating enzyme to induce proliferation and migration of BRCA1-incompetent TNBC cells and TNBC cell lines established from the pleural effusion metastasis of a woman with TNBC. To understand the downstream signaling axis involved in distant metastasis we have used clinically relevant BRCA1 mutant and lung metastatic TNBC cell lines and our results show deregulated expression of caveolin-1, VEGF and SIRT1 in these cells compared to normal mammary epithelial cells by immunofluorescence analysis. We observed SIRT1 to be induced by wild type BRCA1a and BRCA1a I26A mutant unlike the disease associated Ubc9 binding mutants in TNBC cells. Knock down of Ubc9 induced SIRT1 expression in TNBC and ER-α expression in breast cancer cells. This is the first report demonstrating a role for Ubc9 in repressing both SIRT1 and ER-α expression in BRCA1 associated TNBC cells. It also suggests that the BARD-dependent E3 Ubiquitin ligase and HR (homologous recombination) activity of BRCA1 may not be required for inducing SIRT1 expression. Our results suggest for the first time that in BRCA1 mutant TNBC Ubc9-mediated induction of VEGF, inhibition of caveolin-1, SIRT1 and ER-α expression as a novel molecular mechanism underlying TNBC EMT (epithelial mesenchymal transition) leading to lung metastasis with pleural effusion. Drugs that target Ubc9 to both induce SIRT1 and ER-α or using SIRT1 agonists in combination with chemotherapy can be used as a promising targeted therapeutic approach for treating basal-like metastatic BRCA1-linked TNBC thus reducing the mortality in patients with TNBC.

MEK Inhibitor Selumetinib (AZD6244; ARRY-142886) Prevents Lung Metastasis in a Triple-Negative Breast Cancer Xenograft Model.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19-6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial-mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem cell-like characteristics and a CD44(+)CD24(-/low) expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44(+)CD24(-/low) fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent.