Prevention Of Breast Cancer Metastasis Research Articles

Abstract A016: Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches

Abstract Calpain-1 and calpain-2 are heterodimeric calcium-dependent cysteine proteases composed of the catalytic subunits CAPN1 or CAPN2, respectively, and the common regulatory subunit CAPNS1. They are associated with cancer progression, metastasis, and treatment resistance in breast cancer. Here, we present novel insights into the therapeutic potential of calpain inhibition by combining genetic disruption and preclinical mouse tumor models and biosensor-based detection of calpain heterodimerization with ongoing efforts to discover small-molecule and peptide inhibitors. CRISPR-Cas9-mediated knockout of CAPN1, CAPN2, or CAPNS1 in MDA-MB-231 human triple-negative breast cancer (TNBC) cells revealed that disruption of both calpains, through CAPNS1 knockout, significantly reduced cell migration and inhibited spontaneous metastasis in a mouse orthotopic engraftment model by over 80%. Individual knockouts of CAPN1 or CAPN2 also reduced metastasis but to a lesser extent, highlighting the necessity of dual inhibition for optimal therapeutic effect. In parallel, we have been actively seeking small molecules and peptide inhibitors to target calpain through two approaches: inhibiting the PEF-PEF interaction that mediates heterodimerization using small molecules; and targeting the active site using a calpastatin (CAST)-based peptide. To identify small molecules capable of disrupting the PEF-PEF interaction, we conducted in silico screens of over 3.6 million compounds from several libraries. These compounds were evaluated based on their calculated binding affinity and potential to sterically hinder the conformational changes required for calpain activity. The CAST-based peptide inhibitor was designed based on the active site binding B-domain and tested on a purified calpain-2. These efforts lay the groundwork for novel therapeutic approaches targeting calpain-1 and calpain-2, which have emerged as promising targets for preventing metastasis in TNBC. We will present our progress in identifying and validating these small molecules and peptides for further development. Citation Format: Ivan Shapovalov, Pitambar Poudel, Shailesh K. Panday, Danielle Harper, Jung Yeon Min, Yan Gao, Kazem Nouri, Emil Alexov, Peter A. Greer. Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A016

Nuclear localization of APLF facilitates breast cancer metastasis

Most breast cancer deaths result from metastases. We previously reported that DNA repair factor and histone chaperone Aprataxin PNK-like Factor (APLF) is involved in EMT-associated metastasis of triple negative breast cancer (TNBC) cells. However, non-metastatic cells also expressed APLF, the implications of which in disease advancement remain uncertain. Here, we demonstrate that the metastatic prognosis of breast cancer cells may be determined by the cellular localization of APLF. Using TNBC patient samples and cell lines, we discovered that APLF was localized in the nucleus and cytoplasm, whereas other subtypes of breast cancer had cytosolic or perinuclear localization. To investigate metastatic properties in vitro and in vivo, we modeled APLF differential localization by stably producing APLF-tagged nuclear localization signal (NLS) in the luminal subtype MCF7 cells in the absence of putative APLF NLS. Nuclear APLF in non-metastatic MCF7 cells demonstrated pronounced migration, invasion and metastatic potential. We obtained the mechanistic insight from molecular studies that PARP1 could facilitate the transport of APLF from the cytosol to the nucleus, assisting in the metastasis of TNBC cells linked with EMT. Inhibition of PARP1 enzymatic activity with olaparib abrogated the nuclear expression of APLF with loss in expression of genes associated with EMT. Thus, our findings reveal that cellular localization of APLF may predict the risk of breast cancer to metastasize and hence could be exploited to determine the disease progression. We anticipate that the inhibition of cytosolic PARP1-APLF interaction may potentially aid in the prevention of breast cancer metastasis in TNBC patients.

Cysteine protease I29 propeptide from Calotropis procera R. Br. As a potent cathepsin L inhibitor and its suppressive activity in breast cancer metastasis.

Breast cancer metastasis is associated with a poor prognosis and a high rate of mortality. Cathepsin L (CTSL) is a lysosomal cysteine protease that promotes tumor metastasis by degrading the extracellular matrix. Gene set enrichment analysis revealed that CTSL expression was higher in tumorous than in non-tumorous tissues of breast cancer patients and that high-level CTSL expression correlated positively with the epithelial-mesenchymal transition. Therefore, we hypothesized that inhibiting CTSL activity in tumor cells would prevent metastasis. In this study, we characterized the inhibitory activity of SnuCalCpI15, the I29 domain of a CTSL-like cysteine protease from Calotropis procera R. Br., and revealed that the propeptide stereoselectively inhibited CTSL in a reversible slow-binding manner, with an inhibitory constant (Ki) value of 1.38 ± 0.71 nM, indicating its potency as an exogenous inhibitor in anti-cancer therapy. SnuCalCpI15 was localized intracellularly in MDA-MB-231 breast cancer cells and suppressed tumor cell migration and invasion. These results demonstrate the potential of SnuCalCpI15 as a novel agent to prevent breast cancer metastasis.

1α,25-Dihydroxyvitamin D Downregulates Adipocyte Impact on Breast Cancer Cell Migration and Adipokine Release.

Excess adiposity is associated with a higher risk of breast cancer metastasis and mortality. Evidence suggests that dietary vitamin D inhibits breast cancer metastasis. However, the mechanistic link between vitamin D's regulation of adipocyte metabolism and metastasis has not been previously investigated. Therefore, the purpose of these experiments was to examine the effect of the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D), on adipocyte release of bioactive compounds and whether the impact on adipocytes leads to inhibition of breast cancer cell migration, an important step of metastasis. Differentiated 3T3-L1 adipocytes were treated with 1,25(OH)2D for two days, followed by either harvesting the adipocytes or collecting adipocyte-conditioned media without 1,25(OH)2D. A transwell migration assay was conducted with vehicle- or 1,25(OH)2D-conditioned media. In order to explore the mechanism underlying effects on breast cancer metastatic capability, the mRNA expression of leptin, adiponectin, insulin-like growth factor (IGF-1), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) was measured in adipocytes following either vehicle or 1,25(OH)2D treatment. Conditioned media from 1,25(OH)2D-treated adipocytes inhibited the migration of metastatic MDA-MB-231 breast cancer cells compared to conditioned media from vehicle-treated adipocytes. Treatment of adipocytes with 1,25(OH)2D decreased mRNA expression of leptin, adiponectin, IGF-1, IL-6, and MCP-1. Consistent with mRNA expression, concentrations of leptin, adiponectin, IGF-1, and IL-6 in adipocyte-conditioned media were decreased with 1,25(OH)2D treatment, although MCP-1 remained unchanged. In summary, these results suggest that 1,25(OH)2D alters adipocyte secretions to prevent breast cancer metastasis.

Lung-derived soluble factors support stemness/plasticity and metastatic behaviour of breast cancer cells via the FGF2-DACH1 axis

Patients with triple-negative breast cancer (TNBC) have an increased propensity to develop lung metastasis. Our previous studies demonstrated that stem-like ALDHhiCD44+ breast cancer cells interact with lung-derived soluble factors, resulting in enhanced migration and lung metastasis particularly in TNBC models. We have also observed that the presence of a primary TNBC tumor can ‘prime’ the lung microenvironment in preparation for metastasis. In this study, we hypothesized that soluble lung-derived factors secreted in the presence of a primary TNBC tumor can influence stemness/plasticity of breast cancer cells. Using an ex vivo pulmonary metastasis assay (PuMA), we observed that the lung microenvironment supports colonization and growth of ALDHhiCD44+ TNBC cells, potentially via interactions with lung-derived FGF2. Exposure of TNBC cells to lung-conditioned media (LCM) generated from mice bearing TNBC primary tumors (tbLCM) significantly enhanced the proportion of ALDHhiCD44+ cells compared to control or LCM from tumor-naïve mice (tnLCM). Further analysis using a human cancer stem cell qPCR array revealed that, relative to tnLCM or control, exposure of TNBC cells to tbLCM leads to downregulation of the transcription factor and putative tumor suppressor Dachshund homolog 1 (DACH1), a downstream regulator of FGF2. In addition, inhibition of DACH1 using siRNA or treatment with recombinant FGF2 enhanced the ALDHhiCD44+ phenotype. Taken together, our findings suggest that the FGF2-DACH1 signaling axis supports stemness/plasticity of TNBC cells in the lung microenvironment and lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.

Breaking Physical Barrier of Fibrotic Breast Cancer for Photodynamic Immunotherapy by Remodeling Tumor Extracellular Matrix and Reprogramming Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) assist in breast cancer (BRCA) invasion and immune resistance by overproduction of extracellular matrix (ECM). Herein, we develop FPC@S, a photodynamic immunomodulator that targets the ECM, to improve the photodynamic immunotherapy for fibrotic BRCA. FPC@S combines a tumor ECM-targeting peptide, a photosensitizer (protoporphyrin IX) and an antifibrotic drug (SIS3). After anchoring to the ECM, FPC@S causes ECM remodeling and BRCA cell death by generating reactive oxygen species (ROS) in situ. Interestingly, the ROS-mediated ECM remodeling can normalize the tumor blood vessel to improve hypoxia and in turn facilitate more ROS production. Besides, upon the acidic tumor microenvironment, FPC@S will release SIS3 for reprograming CAFs to reduce their activity but not kill them, thus inhibiting fibrosis while preventing BRCA metastasis. The natural physical barrier formed by the dense ECM is consequently eliminated in fibrotic BRCA, allowing the drugs and immune cells to penetrate deep into tumors and have better efficacy. Furthermore, FPC@S can stimulate the immune system and effectively suppress primary, distant and metastatic tumors by combining with immune checkpoint blockade therapy. This study provides different insights for the development of fibrotic tumor targeted delivery systems and exploration of synergistic immunotherapeutic mechanisms against aggressive BRCA.

Simultaneous Visualization and Depletion of Peroxynitrite by a Simple Aggregation-Induced Emission Nanoprobe for Preventing Breast Cancer Metastasis after Surgery.

Inflammation has been confirmed to be closely related to the development of tumors, while peroxynitrite (ONOO-) is one of the most powerful oxidative pro-inflammatory factors. Although ONOO- can kill bacteria through oxidation, it will activate matrix metalloproteinases (MMPs), accelerate the degradation of the extracellular matrix (ECM), and subsequently lead to the activation and release of other tumor promotion factors existing in the ECM, promoting tumor metastasis and invasion. Herein, we report a simple aggregation-induced emission (AIE) nanoprobe (NP), TPE-4NMB, that can simultaneously visualize and deplete ONOO-. The probe can light up the endogenous and exogenous ONOO- in cells and selectively inhibit the proliferation and migration of 4T1 cells by inducing an intracellular redox homeostasis imbalance through ONOO- depletion. After being modified with DSPE-PEG2000, the TPE-4NMB NPs can be used to image ONOO- induced by various models in vivo; especially, it can monitor the dynamic changes of ONOO- level in the residual tumor after surgery, which can provide evidence for clarifying the association between surgery, ONOO-, and cancer metastasis. Excitingly, inhibited tumor volume growth and decreased counts of lung metastases were observed in the TPE-4NMB NPs group, which can be attributed to the downregulated expression of MMP-9 and transforming growth factor-β (TGF-β), increased cell apoptosis, and inhibited epithelial-mesenchymal transition (EMT) mediated by ONOO-. The results will provide new evidence for clarifying the relationship between surgery, ONOO-, and tumor metastasis and serve as a new intervention strategy for preventing tumor metastasis after tumor resection.

Ganoderma lucidum spore oil synergistically enhances the function of cyclophosphamide in the prevention of breast cancer metastasis.

Ganoderma lucidum ( G . lucidum ) is a traditional Chinese herbal medicine that has shown potential as an alternative adjuvant therapy for cancer patients. However, the mechanisms and adjuvant therapeutic effects of G . lucidum in cancer treatment remain unclear. In this work, G . lucidum spore oil (GanoOil), a newly developed oily G . lucidum spore extract was used to investigate the mechanisms and adjuvant therapeutic effects of GanoOil in conjunction with the chemotherapeutic drug cyclophosphamide (CTX) for preventing breast cancer metastasis. In the model of lung metastasis, orally administered GanoOil increased the population of CD8 + T cells and interleukin (IL)-6 cytokine levels in mouse blood, whereas also enhancing the activity of natural killer cells in the spleen. Furthermore, the combination of GanoOil and CTX effectively suppressed the lung metastasis of circulating breast cancer cells, alleviated CTX-induced weight loss, and reduced the ratio of lung and spleen weight to body weight in mice. Moreover, high concentrations of GanoOil exhibited no significant toxicity or side effects in both in vitro and in vivo experiments. In conclusion, GanoOil is a safe drug that can enhance immune activity in mice to achieve therapeutic effects on cancer, and can also synergistically inhibit tumor metastasis with CTX.

PAR2 deficiency tunes inflammatory microenvironment to magnify STING signalling for mitigating cancer metastasis via anionic CRISPR/Cas9 nanoparticles

Metastasis is one of the most significant causes for deterioration of breast cancer, contributing to the clinical failure of anti-tumour drugs. Excessive inflammatory responses intensively promote the occurrence and development of tumour, while protease-activated receptor 2 (PAR2) as a cell membrane receptor actively participates in both tumour cell functions and inflammatory responses. However, rare investigations linked PAR2-mediated inflammatory environment to tumour progression. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is an emerging and powerful gene editing technique and can be applied for probing the new role of PAR2 in breast cancer metastasis, but it still needs the development of an efficient and safe delivery system. This work constructed anionic bovine serum albumin (BSA) nanoparticles to encapsulate CRISPR/Cas9 plasmid encoding PAR2 sgRNA and Cas9 (tBSA/Cas9-PAR2) for triggering PAR2 deficiency. tBSA/Cas9-PAR2 remarkably promoted CRISPR/Cas9 to enter and transfect both inflammatory and cancer cells, initiating precise PAR2 gene editing in vitro and in vivo. PAR2 deficiency by tBSA/Cas9-PAR2 effectively suppressed NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signalling in inflammatory microenvironment to magnify stimulator of interferon genes (STING) signalling, reactive oxygen species (ROS) accumulation and epithelial-mesenchymal transition (EMT) reversal, consequently preventing breast cancer metastasis. Therefore, this study not only demonstrated the involvement and underlying mechanism of PAR2 in tumour progression via modulating inflammatory microenvironment, but also suggested PAR2 deficiency by tBSA/Cas9-PAR2 as an attractive therapeutic strategy candidate for breast cancer metastasis.

Identification of Breast Cancer Metastasis Markers from Gene Expression Profiles Using Machine Learning Approaches.

Cancer metastasis accounts for approximately 90% of cancer deaths, and elucidating markers in metastasis is the first step in its prevention. To characterize metastasis marker genes (MGs) of breast cancer, XGBoost models that classify metastasis status were trained with gene expression profiles from TCGA. Then, a metastasis score (MS) was assigned to each gene by calculating the inner product between the feature importance and the AUC performance of the models. As a result, 54, 202, and 357 genes with the highest MS were characterized as MGs by empirical p-value cutoffs of 0.001, 0.005, and 0.01, respectively. The three sets of MGs were compared with those from existing metastasis marker databases, which provided significant results in most comparisons (p-value < 0.05). They were also significantly enriched in biological processes associated with breast cancer metastasis. The three MGs, SPPL2C, KRT23, and RGS7, showed highly significant results (p-value < 0.01) in the survival analysis. The MGs that could not be identified by statistical analysis (e.g., GOLM1, ELAVL1, UBP1, and AZGP1), as well as the MGs with the highest MS (e.g., ZNF676, FAM163B, LDOC2, IRF1, and STK40), were verified via the literature. Additionally, we checked how close the MGs were to each other in the protein-protein interaction networks. We expect that the characterized markers will help understand and prevent breast cancer metastasis.

Liposome-encapsulated zoledronate increases inflammatory macrophage population in TNBC tumours

BackgroundTumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. MethodsTriple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. ResultsLiposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. ConclusionResults show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.

In Silico Insight to Identify Potential Inhibitors of BUB1B from Mushroom Bioactive Compounds to Prevent Breast Cancer Metastasis.

Breast cancer is one of the most common types of cancer among women worldwide, and its metastasis is a significant cause of mortality. Therefore, identifying potential inhibitors of proteins involved in breast cancer metastasis is crucial for developing effective therapies. BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is a key regulator of mitotic checkpoint control, which ensures the proper segregation of chromosomes during cell division. Dysregulation of BUB1B has been linked to a variety of human diseases, including breast cancer. Overexpression of BUB1B has been observed in various cancer types, and its inhibition has been shown to induce cancer cell death. Additionally, BUB1B inhibition has been suggested as a potential strategy for overcoming resistance to chemotherapy and radiation therapy. Given the importance of BUB1B in regulating cell division and its potential as a therapeutic target, the development of BUB1B inhibitors has been the focus of intense research efforts. Despite these efforts, few small molecule inhibitors of BUB1B have been identified, highlighting the need for further research in this area. In this study, the authors aimed to identify potential inhibitors of BUB1B from mushroom bioactive compounds using computational methods, which could ultimately lead to the development of new treatments for breast cancer metastasis. This study has incorporated 70 bioactive compounds (handpicked through literature mining) of distinct mushrooms that were considered and explored to identify a suitable drug candidate. Their absorption, distribution, metabolism and excretion (ADME) properties were obtained to predict the drug-likeness of these 70 mushroom compounds based on Lipinski's rule of 5 (RO5). Screening these bioactive compounds and subsequent molecular docking against BUB1B provided compounds with the best conformation-based binding affinity. The best two complexes, i.e., BUB1B-lepitaprocerin D and BUB1B-peptidoglycan, were subjected to molecular dynamic simulations. Both complexes were assessed for their affinity, stability, and flexibility in protein-ligand complex systems. The molecular dynamic (MD) simulation studies revealed that lepitaprocerin D has an energetically favorable binding affinity with BUB1B. Results showed that the formation of a hydrogen bond between residues ASN123 and SER157, and lepitaprocerin D had strengthened the affinity of lepitaprocerin D with BUB1B. This study identified lepitaprocerin D as a potential and novel inhibitor for BUB1B that could be a plausible drug candidate for identifying and controlling the spread of breast cancer metastasis.

Investigation of L-carnitine effects on CD44+ cancer stem cells from MDA-MB-231 breast cancer cell line as anti-cancer therapy

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells, capable of metastasis, recurrence, and drug resistance in breast cancer patients. Therefore, targeting BCSCs appears to be a promising strategy for the treatment and prevention of breast cancer metastasis. Mounting evidence supports the fact that carnitine, a potent antioxidant, modulates various mechanisms by enhancing cellular respiration, inducing apoptosis, and reducing proliferation and inflammatory responses in tumor cells. The objective of this study was to investigate the impact of L-carnitine (LC) on the rate of proliferation and induction of apoptosis in CD44+ CSCs. To achieve this, the CD44+ cells were enriched using the Magnetic-activated cell sorting (MACS) isolation method, followed by treatment with LC at various concentrations. Flow cytometry analysis was used to determine cell apoptosis and proliferation, and western blotting was performed to detect the expression levels of proteins. Treatment with LC resulted in a significant decrease in the levels of p-JAK2, p-STAT3, Leptin receptor, and components of the leptin pathway. Moreover, CD44+ CSCs-treated cells with LC exhibited a reduction in the proliferation rate, accompanied by an increase in the percentage of apoptotic cells. Hence, it was concluded that LC could potentially influence the proliferation and apoptosis of CD44+ CSC by modulating the expression levels of specific protein.

Rac and Cdc42 inhibitors reduce macrophage function in breast cancer preclinical models.

Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion and modulation of the tumor microenvironment (TME). A viable anti-metastasis strategy is to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, we tested the hypothesis that Rac and Cdc42 inhibitors target immunosuppressive immune cells, in addition to cancer cells. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. The potential of Rac/Cdc42 inhibitors EHop-016 and MBQ-167 to target macrophages was tested in human and mouse macrophage cell lines via activity assays, MTT assays, wound healing, ELISA assays, and phagocytosis assays. Immunofluorescence, immunohistochemistry, and flow cytometry were used to identify myeloid cell subsets from tumors and spleens of mice following EHop-016 or MBQ-167 treatment. EHop-016 and MBQ-167 inhibited Rac and Cdc42 activation, actin cytoskeletal extensions, migration, and phagocytosis without affecting macrophage cell viability. Rac/Cdc42 inhibitors also reduced tumor- infiltrating macrophages and neutrophils in tumors of mice treated with EHop-016, and macrophages and MDSCs from spleens and tumors of mice with breast cancer, including activated macrophages and monocytes, following MBQ-167 treatment. Mice with breast tumors treated with EHop-016 significantly decreased the proinflammatory cytokine Interleukin-6 (IL-6) from plasma and the TME. This was confirmed from splenocytes treated with lipopolysaccharide (LPS) where EHop-016 or MBQ-167 reduced IL-6 secretion in response to LPS. Rac/Cdc42 inhibition induces an antitumor environment via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells in the TME.

Abstract B011: Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis

Abstract Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion, modulation of the tumor microenvironment (TME), plasticity, and ability to colonize secondary tissues. Therefore, a viable anti-metastasis strategy needs to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, the purpose of this study was to test the hypothesis that Rac and Cdc42 inhibitors target both migratory cancer cells and immunosuppressive cells in the TME. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Since the targets of these inhibitors may also affect immune cell behavior, we first tested their potential to target Rac/Cdc42 function in human and rat macrophage cell lines and show that EHop-016 and MBQ-167 inhibit Rac activation (at 5X higher concentration than the IC50 in breast cancer cells), actin cytoskeletal extensions, and migration, without affecting cell viability. EHop-016 and MBQ-167 significantly decrease the proinflammatory cytokine Interleukin-6 (Il-6) from plasma and the TME. Next, we tested the in vivo effect of MBQ-167 on immune cells from breast cancer mouse models. Tumors from immunodeficient mice bearing mammary tumors that were treated with vehicle or 50mg/kg MBQ-167 5x a week for 5 weeks, were analyzed by flow cytometry to quantify different immune cell types. MBQ-167 treated mice demonstrated elevated CD11+F4/80+CD86+ M1 macrophages and reduced MDSCs in mammary tumors. In the immunocompetent 4T-1 breast cancer model, spleens contained a lower frequency of macrophages and neutrophils, accompanied by an increase in activated cytotoxic CD69+, CD8+T cells. Moreover, when metastasis to the lungs was reduced by ~90% following MBQ-167 treatment, granzyme B+ CD8+ T cells were elevated in the lung extracts, without affecting the immunosuppressive CD4+ regulatory T cell counts. In vitro, we also demonstrated that increasing doses of MBQ-167 induced T cell activation and interferon-gamma secretion, a cytokine known to have antitumor properties. Taken together, Rac/Cdc42 inhibitors induce anti-metastatic cancer effects via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells, while promoting the activation of cytotoxic T cells, in the tumor and the metastasis microenvironment. Therefore, MBQ-167 promises to be an effective antimetastatic cancer therapeutic that is poised to enter Phase I clinical trials. Citation Format: Stephanie Dorta-Estremera, Anamaris Torres, Michael Rivera-Robles, Ailed Cruz-Collazo, Luis Borrero-Garcia, Suranganie Dharmawardhane. Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B011.

SIRT7 Downregulation Promotes Breast Cancer Metastasis Via LAP2α-Induced Chromosomal Instability.

Chromosomal instability (CIN) plays an important role in the initiation and progression of carcinomas. However, the regulatory mechanism of metastasis mediated by CIN in breast cancer is not fully understood. Here, we aimed to demonstrate that the deregulation of SIRT7 and lamina-associated polypeptide 2α (LAP2α) critically contributes to CIN-induced metastasis in breast cancer. Expression of SIRT7 and chromosome stability-related genes was examined using western blotting, quantitative real-time PCR, immunohistochemistry, and immunofluorescence; functional significance of SIRT7 was examined using in vitro and in vivo models; and interaction between SIRT7 and LAP2α was assessed by co-inmunoprecipitation (Co-IP) assays. Doxorubicin (DOX) inhibited SIRT7 expression and enhanced CIN in breast cancer cells; SIRT7 deficiency led to CIN in breast cancer cells. Co-IP approach and immunohistochemistry demonstrated that SIRT7 interacted directly and positively with LAP2α and SIRT7 knockdown led to increased ubiquitination-dependent degradation of LAP2α and reduced protein levels of LAP2α, whereas LAP2α knockdown did not affect SIRT7 expression. In vitro and in vivo evidence revealed that SIRT7 promotes breast cancer metastasis through the SIRT7/LAP2α axis. In summary, SIRT7 interacts with LAP2α to regulate CIN and metastasis in breast cancer, and inhibition of SIRT7/LAP2α axis represents a potential therapeutic strategy for preventing breast cancer metastasis.

Evaluation of the Lysyl Oxidase-Like 2 (LOXL2) inhibitory activity of pimaranes and their glycosyl derivatives

Lysyl oxidase (LOX) and LOX-like 1-4 (LOXL 1-4) enzymes catalyze the cross-linking of elastin and collagen in the extracellular matrix, facilitating cell migration and invasion. The inhibition of these enzymes, particularly LOXL2, has been suggested as a therapeutic strategy to prevent breast cancer metastasis. In this work, new natural LOXL2 inhibitors were searched from Aeollanthus rydingianus, a medicinal plant rich in bioactive products. Five pimarane diterpenoids, two isolated from the plant and three derivatives, were tested. These compounds have been described for their bioactive properties such as anti-tumor, anti-inflammatory, analgesic, and antibacterial activities. In this regard, we intended to explore the mechanisms of these compounds by studying their effects on LOXL2 activity. Two pimarane diterpenoids showed a mild LOXL2 inhibitory activity as evaluated by an Amplex Ultra Red-based technique. The cytotoxicity of the most active compound was analyzed by the MTT assay in the MDA-MB-231 cell line, representative of triple-negative breast cancer. This compound decreased cell viability as single agent and increased the cytotoxic effect of doxorubicin. Its glycoconjugate was considerably more toxic, likely due to a higher uptake by cancer cells. Keywords: breast cancer, lysyl oxidase-like 2, inhibitors, pimaranes, Aeollanthus rydingianus, MTT

Potent and Selective Inhibition of CYP1A2 Enzyme by Obtusifolin and Its Chemopreventive Effects.

Obtusifolin, a major anthraquinone component present in the seeds of Cassia tora, exhibits several biological activities, including the amelioration of memory impairment, prevention of breast cancer metastasis, and reduction of cartilage damage in osteoarthritis. We aimed to evaluate the inhibitory effects of obtusifolin and its analogs on CYP1A enzymes, which are responsible for activating procarcinogens, and investigate its inhibitory mechanism and chemopreventive effects. P450-selective substrates were incubated with human liver microsomes (HLMs) or recombinant CYP1A1 and CYP1A2 in the presence of obtusifolin and its four analogs. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. Molecular docking simulations were performed using the crystal structure of CYP1A2 to identify the critical interactions between anthraquinones and human CYP1A2. Obtusifolin potently and selectively inhibited CYP1A2-mediated phenacetin O-deethylation (POD) with a Ki value of 0.031 µM in a competitive inhibitory manner in HLMs, whereas it exhibited negligible inhibitory effect against other P450s (IC50 > 28.6 µM). Obtusifolin also inhibited CYP1A1- and CYP1A2-mediated POD and ethoxyresorufin O-deethylation with IC50 values of <0.57 µM when using recombinant enzymes. Our molecular docking models suggested that the high CYP1A2 inhibitory activity of obtusifolin may be attributed to the combination of hydrophobic interactions and hydrogen bonding. This is the first report of selective and potent inhibitory effects of obtusifolin against CYP1A, indicating their potential chemopreventive effects.

Blockade of Platelet CysLT1R Receptor with Zafirlukast Counteracts Platelet Protumoral Action and Prevents Breast Cancer Metastasis to Bone and Lung.

Metastases are the main cause of death in cancer patients, and platelets are largely known for their contribution in cancer progression. However, targeting platelets is highly challenging given their paramount function in hemostasis. Using a high-throughput screening and platelet-induced breast tumor cell survival (PITCS) assay as endpoint, we identified the widely used anti-asthmatic drugs and cysteinyl leukotriene receptor 1 (CysLT1R) antagonists, zafirlukast and montelukast, as new specific blockers of platelet protumoral action. Here, we show that human MDA-B02 breast cancer cells produce CysLT through mechanisms involving microsomal glutathione-S-transferase 1/2/3 (MGST1/2/3) and that can modulate cancer cell–platelet interactions via platelet–CysLT1R. CysLT1R blockade with zafirlukast decreased platelet aggregation and adhesion on cancer cells and inhibited PITCS, migration, and invasion in vitro. Zafirlukast significantly reduced, by 90%, MDA-B02 cell dissemination to bone in nude mice and reduced by 88% 4T1 spontaneous lung metastasis formation without affecting primary tumor growth. Combined treatment of zafirlukast plus paclitaxel totally inhibited metastasis of 4T1 cells to the lungs. Altogether, our results reveal a novel pathway mediating the crosstalk between cancer cells and platelets and indicate that platelet CysLT1R represents a novel therapeutic target to prevent metastasis without affecting hemostasis.

Fucoxanthin prevents breast cancer metastasis by interrupting circulating tumor cells adhesion and transendothelial migration.

Metastasis is the leading cause of cancer-related death and a critical challenge in improving cancer treatment today. Circulating tumor cells (CTCs) adhesion to and across the vascular endothelium are critical steps in the establishment of micrometastatic foci away from the primary tumor. Therefore, we believe that interrupting CTCs adhesion to endothelium and transendothelial migration may efficiently prevent cancer metastasis. Fucoxanthin (Fx) is an algal carotenoid widely distributed in brown algae, macroalgae, and diatoms. Previous studies have found that Fx has various pharmacological activities, including antidiabetic, antioxidant, anti-inflammatory, anti-obesity, antimalarial, anticancer, and so on. However, it remains unclear whether Fx has a preventive effect on cancer metastasis. Here, we found that Fx interrupts breast cancer cells MCF-7 adhesion to endothelium and transendothelial migration, thus inhibiting CTCs-based pulmonary metastasis in vivo. The hetero-adhesion assay showed that Fx significantly inhibited the expression of inflammatory factor-induced cell adhesion molecules (CAMs) and the resulting adhesion between MCF-7 cells and endothelial cells. The wound-healing and transwell assays showed that Fx significantly inhibited the motility, invasion, and transendothelial migration abilities of MCF-7 cells. However, the same concentration of Fx did not significantly alter the cell viability, cell cycle, apoptosis, and ROS of breast cancer cells, thus excluding the possibility that Fx inhibits MCF-7 cell adhesion and transendothelial migration through cytotoxicity. Mechanistically, Fx inhibits the expression of CAMs on endothelial cells by inhibiting the NF-кB signaling pathway by down-regulating the phosphorylation level of IKK-α/β, IкB-α, and NF-кB p65. Fx inhibits transendothelial migration of MCF-7 cells by inhibiting Epithelial-to-mesenchymal transition (EMT), PI3K/AKT, and FAK/Paxillin signaling pathways. Moreover, we demonstrated that Fx significantly inhibits the formation of lung micrometastatic foci in immunocompetent syngeneic mouse breast cancer metastasis models. We also showed that Fx enhances antitumor immune responses by substantially increasing the subsets of cytotoxic T lymphocytes in the peripheral immune system. This new finding provides a basis for the application of Fx in cancer metastatic chemoprevention and suggests that interruption of the CTCs adhesion to endothelium and transendothelial migration may serve as a new avenue for cancer metastatic chemoprevention.