Cannabinoids and triple-negative breast cancer treatment

Abstract

1. Triple-negative breast cancer

Triple-negative breast cancer (TNBC) accounts for about 10-20% of all breast cancer cases and is associated with the worst prognosis. TNBC is a heterogeneous group of tumors defined by the absence of the estrogen receptor (ER), the progesterone receptor (PR), and the absence of the human epidermal growth factor receptor 2 (HER2). It mainly occurs in younger patients and patients with BRCA germline mutations (1, 2). TNBC has been divided into six different subgroups: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM) and luminal androgen receptor (LAR) (3). The TNBC microenvironment (TME) includes tumor-infiltrating lymphocytes (TILs) with CD3+ T and CD20+ B lymphocytes, CD38+/CD138+ plasma cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs), natural killer (NK) cells and cancer-associated adipocytes (CAAs). The TNBC TME is unique in that it has a high expression of vascular endothelial growth factors and a high infiltration rate of TILs and TAM and plays an important role in the pathogenesis of the disease. Type M2 macrophages are upregulated compared to other subtypes of breast cancer. CAAs also play a greater role. A variety of cytokines and chemokines influence tumor growth, metastasis and drug resistance, mediate immunosuppression and antitumor activity, and play an important role in TNBC TME (4). Dendritic cells (DCs) are antigen-presenting cells (APCs) that play a crucial role in acquired and innate immune responses and are involved in the development of T cell-mediated antitumor immune responses (5). Neoangiogenesis is necessary for the nutrition and oxygen supply of the tumor and thus for the progression of the tumor. The endothelium is involved in the invasion of tumor cells into the vascular lumen and the resulting metastasis. In addition, endothelial cells are one of the main sources of CAFs (6). Until recently, treatment options for TNBC were limited to chemotherapy, e.g. anthracyclines and taxanes. The prognosis of metastatic TNBC is poor, with a median survival of 11 to 17 months. A new successful systemic treatment is immunotherapy with inhibitors of programmed cell death ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1), also known as immune checkpoint inhibitors (ICI). Atezolizumab (PD-L1 inhibitor) and pembrolizumab (PD-1 inhibitor) are in clinical use (7). Atezolizumab is used for the treatment of metastatic or unresectable locally advanced breast cancer and is restricted to patients whose tumors express PD-L1 on immune cells (> 1% PD-L1 positive tumor cells) (8). Pembrolizumab is used in early-stage TNBC (9) and in patients with advanced cancer with a combined positive score (CPS score), defined as the number of PD-L1–staining cells (tumor cells, lymphocytes and macrophages) divided by the total number of viable tumor cells multiplied by 100) > 10 (10). In TNBC PD-1/PD-L1 are used as biomarkers for response to immunotherapy. However, new tumor-specific biomarkers are needed to improve patient outcomes (11). In patients with BRCA mutation, poly (ADP-ribose) polymerase (PARP) inhibitors are used in adjuvant and metastatic treatment. This is an example of synthetic lethality and targeted treatment (12, 13). Newer treatment options are the antibody-drug conjugates sacituzumab govitecan and trastuzumab deruxtecan. Sacituzumab govitecan is an antibody directed against Trop2 to which SN38 (a derivative of irinotecan) is bound (14). Trastuzumab deruxtecan is an antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab covalently linked to the topoisomerase I inhibitor deruxtecan (a derivative of exatecan). It is used in a subset of TNBC that is considered HER2-low, a category that blurs the distinction between HER2-positive and TNBC (15).

2. Cannabinoids and the endocannabinoid system

Cannabinoid receptors (CBRs) are membrane-bound G protein-coupled receptors (GPCRs) and were identified over 30 years ago. Cannabinoid receptor 1 (CB1R) is one of the most abundant receptors in the brain. To a lesser extent, CB1R is also expressed in the periphery, including the immune system. Cannabinoid receptor 2 (CB2R) is also expressed in the central nervous system, but to a much lesser extent than CB1R. In addition, CB2R is highly expressed in the periphery, particularly in organs that are part of the immune system and in other peripheral tissues. Natural polymorphisms and alternative splice variants may be important for its function (16). In addition, there is a “non-canonical” extended signaling network of the endocannabinoid system. It consists of fatty acid derivatives, ionotropic cannabinoid receptors (transient receptor potential (TRP) channels) and other GPCRs (GPR18, GPR19, GPR55, peroxisome proliferator-activated receptor alpha (PPARα), eCB), enzymes involved in the biosynthesis and degradation of endocannabinoids (fatty-acid amide hydrolase 1 (FAAH) and monoacylglycerol lipase (MAGL)), and protein transporters fatty-acid-binding proteins (FABPs)) (17–22). Cannabinoid receptors recognize different agonists and antagonists and can be activated by endogenous or exogenous cannabinoids. Endogenous cannabinoids or endocannabinoids are metabolites of arachidonic acid (AA). The best studied are N-arachidonoylethanolamine (anandamide, the Sanskrit word for “bliss”) and 2-arachidonoylglycerol (2-AG), which together with the CBRs form the endocannabinoid system (23). Anandamide binds to CBRs with higher affinity than 2-AG, but acts only as a partial agonist (24). 2-AG, on the other hand, behaves as a full agonist for CBRs (25, 26). 2-AG binds selectively to CBRs, whereas anandamide is less selective (27). It has been suggested that 2-AG and not anandamide is the actual natural ligand for CBRs (28–30). Tetrahydrocannabinol (THC) is the main psychoactive constituent of Cannabis sativa and thus an exogenous phytocannabinoid and a non-selective agonist of CB1R and CB2R (31). Other important phytocannabinoids are cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), cannabidivarin (CBDV) and tetrahydrocannabivarin (THCV) (32). Compared to THC, CBD has a lower CB1R and CB2R affinity and acts as an inverse agonist on CB2R. It is a non-psychoactive substance and therefore a potential therapeutic agent (33). Synthetic cannabinoids (i.e. JWH-015, WIN55,212-2, compound-10, etc.) are a heterogeneous group of substances that can be selective agonists or antagonists of CB1R or CB2R (34). CB1R and CB2R exhibit allosteric binding and biased signaling that influences the biological response mediated by agonists (31, 35).

3. Cannabinoids and the immune system

Various components of the endocannabinoid system act as important regulators of the immune system and the immune response (36–38). Exogenous cannabinoids are generally immunosuppressive and potent immunological mediators (39–43). Immune cells express both CB1R and CB2R (44, 45), although the expression of CB1R is significantly lower compared to CB2R (41, 46). CB2R is mainly found in cells of the immune system and plays an important role as a modulator of immune function (47–49). The activation of CB2R in many immune cells is central to the suppressive effects of cannabinoids. However, the pro-inflammatory effects appear to be linked to the expression of CB1R. Cannabinoids inhibit adenylyl cyclase and activate beta-gamma-mediated signaling pathways and modulate intracellular free calcium levels. These changes may negatively affect the release of inflammatory mediators and the induction of pro-inflammatory transcriptional programs. Exposure to cannabinoids inhibits the release of prostaglandins, histamine, and matrix-active proteases from mast cells (50). Phagocytic function is suppressed by cannabinoids. Cannabinoids also suppress inflammation at a secondary level by downregulating the production of cytokines (e.g. tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin-1 (IL-1), and IL-4 (41, 51–54). There are relatively few descriptions of immunological side effects of cannabinoids in humans. However, the human immune system is altered by chronic exposure to cannabinoids (55). Cannabinoid exposure does not accelerate the loss of immunocompetence in HIV-1 infected patients (56, 57). Cannabis use is associated with long-term changes in immunological homeostasis (58, 59). Inhibition of cell-mediated immunity has been found in marijuana smokers (60) and cannabis smoking causes inflammatory changes in the airways (61). Studies have shown that exposure to cannabinoids leads to a suppression of responsiveness to infectious diseases, and a link between cannabis use and increased susceptibility to various infections has been suggested (62–64). These studies suggest a significant potential for immunological side effects of cannabinoid compounds in humans. Interestingly, cannabinoids can affect the gut microbiota (65–67), which also influences immunotherapy for cancer (68–71).

4. Cells of the TNBC microenvironment and the endocannabinoid system

It appears that most of the cell types present in the TNBC TME express CBRs and may be affected in some way by the exogenous cannabinoids and/or the endocannabinoid system (4, 72). CB2R is expressed in CD8+ T lymphocytes and CD4+ T lymphocytes and CB1R mRNA transcripts are modestly present in human T- lymphocytes (40, 41, 73). The expression of the CB2R receptor is low in circulating T lymphocytes. However, several studies have reported that CB2R receptor expression is increased in activated T lymphocytes and that its activation decreases their proliferation (74–76). This is associated with reduced production of pro-inflammatory cytokines and increased apoptosis (74–78). In addition, CBR agonists can upregulate immunosuppressive cytokines (79, 80) and cause inhibition of chemotaxis (81–83). The antitumor function of T lymphocytes is enhanced in Cnr2 conditional knockout mouse (84). The activation of CB2R appears to have different effects depending on the subtype of T lymphocytes, with the functions of Th1 and Th17 tending to be reduced and those of Th2 promoted in humans (72, 75, 85, 86). In addition, the CB2R receptor is highly expressed in cytokine-induced killer (CIK) cells, a subset of cytotoxic T lymphocytes with a CD3+ CD56+ immunophenotype (4, 87–90). It is important to note that the activation of T lymphocytes is the primary mechanism of action of ICI (91, 92). B-cells express CB1R and CB2R (93, 94). Treatment with a CB2R agonist increased the proliferation of B lymphocytes, a phenomenon that was blocked by a CB2R antagonist (95). In mice, activation of the CB2R receptor was associated with differentiation, migration, proliferation and antibody class switching of B lymphocytes (96–98). These results suggest that CB2R is part of the immune programming of B lymphocytes and plays an important role in the development of B lymphocytes (72, 99). CBRs are overexpressed in chronic lymphocytic leukemia (CLL) compared to healthy B lymphocytes and CBR1 could be a new prognostic marker (100). CB2R is expressed in monocytes and CB1R mRNA transcripts are modestly present (40, 41, 73). CB2R agonists can modulate human monocyte migration (101, 102). Macrophages express both CB1R and CB2R (103, 104). Several studies have shown that cannabinoids negatively regulate phagocytosis, cell-spreading, and antigen presentation by macrophages (105–107). CB2R has been shown to switch the polarization of M1 macrophages to M2 macrophages (108–110). CB2R is expressed in human fibroblasts (111) and transcripts for CB1R and CB2R have been found in both odontoblasts and gingival fibroblasts (112). Fibroblasts also express enzymes that metabolize endocannabinoids. Fibroblasts can be influenced by autocrine signaling of endocannabinoids via CB1R and CB2R and paracrine signaling by neighboring leukocytes (113). CB2R is expressed in polymorphonuclear neutrophils (40, 41, 73). Cannabinoids can influence neutrophil function via CBRs or other mechanisms. The inhibitory effect of cannabinoids on neutrophil functional responses is mostly related to a mechanism other than CBRs, consistent with the absence or very low expression of CB2R (114–119). Neutrophil migration is related to the expression of CB2R and GPR55 (120). Chemotaxis of human neutrophils is inhibited by CBR agonists (114, 121). CB1R and CB2R are expressed in NK cells. The predominance of CB2R in NK cells is illustrated by a striking ratio of 100:1 between CB2R and CB1R (40, 41, 44, 73, 122–125). NK cells release large amounts of endocannabinoids (125) and endocannabinoids influence the chemotaxis of NK cells (126). NK cell activity is inhibited by CBR agonists (127), and cannabis use has been associated with a decrease in NK cell counts (58, 59). Mature adipocytes from visceral and subcutaneous adipose tissue express CBRs on their plasma membranes and CB1R is located at various subcellular levels, including the plasma membrane and mitochondria of the adipocyte (128, 129). However, there is some uncertainty regarding CB2R expression in differentiated adipocytes (130–134). A complete endocannabinoid system has been found in both murine and human adipocytes (135–137). Bone marrow-derived DCs from mice express CB1R and CB2R (138) and the endocannabinoid system is present in human DCs (139). Endocannabinoids act as chemoattractants for DCs and activation of CBRs induces apoptosis (138, 140). Both CB1R and CB2R are expressed in endothelial cells (141, 142). It is possible that an atypical cannabinoid receptor, the endothelial cannabinoid receptor (eCB receptor), is responsible for the vasodilatory effect of cannabinoids (143). A study found that CBRs are expressed in glioblastoma endothelial cells. CB1R expression was detected in about 38% and CB2R expression in 54% of cells. Compared to CB1R, the expression of CB2R was increased in the endothelial cells of glioblastoma (142).

5. Cannabinoid receptor expression in TNBC

The presence of CBRs in human breast tumors was investigated by quantitative real-time PCR and confocal microscopy. Lower levels of CB1R mRNA were detected in low, intermediate and high histologic grade tumors compared to normal, non-cancerous breast tissue. In all tumors examined, CB2R expression was higher than CB1R expression. Hormone receptor-negative (HR-) tumors expressed more CB2R mRNA than ER +/PR + tumors (144). Perez-Gomez et al. performed a histopathologic analysis of tissue samples for the expression of CBRs. A very large proportion of human breast adenocarcinomas (~75%) expressed CB2R and expression was strongly associated with HER2+ tumors, while no association was found between CB2R expression and HR+ or TNBC. There was an association between increased expression of CB2R and poorer prognosis and higher likelihood of local recurrence in HER2+ breast cancer (145). The orphan receptor GRP55 could play an important role in TNBC. In the study by Andradas et al. it was found that the TNBC cell line MDA-MB-231 reduced its invasive behavior when GPR55 expression was knocked down. Similar effects were observed in an animal model of lung metastasis. In breast cancer patients, there is a strong association between GPR55 protein levels and TNBC tumors. Using tissue microarrays and publicly available data from The Cancer Genome Atlas (TCGA), it has been shown that higher GPR55 expression is associated with poorer patient prognosis (lower disease-free survival and metastasis-free survival) (146).

6. Effect of different cannabinoids on TNBC

The antitumor effect of THC on breast cancer cell lines was documented. Among the tumor cells, those with a more aggressive phenotype, including the MDA-MB-231 cell line, were more sensitive to THC. One mechanism of this effect is that THC arrests the cells at the G₂-M cell cycle checkpoint via downregulation of the cyclin-dependent kinase 1 (Cdc2), as suggested by the reduced sensitivity of Cdc2-overexpressing cells to THC (144). In addition, CBD has an antitumor and anti-metastatic effect in TNBC cell lines. The antitumor effect of CBD is mediated by the activation of apoptosis. Apoptosis in the MDA-MB-231 cell line can be induced by direct or indirect activation of the CB2R receptor and transient receptor potential vanilloid type-1 (TRPV1) as well as by an increase in intracellular Ca²⁺ and reactive oxygen species (ROS) independent of the cannabinoid/vanilloid receptors (147). CBD induces an interaction between the PPARγ, the mammalian target of rapamycin (mTOR) and cyclin D1, which promotes apoptosis (148) and can downregulate the expression of an inhibitor of basic helix-loop-helix transcription factors (Id-1) in metastatic TNBC cells, which leads to a reduction in tumor aggressiveness (149). The downregulation of Id-1 expression is the result of differential modulation of extracellular signal-regulated kinase (ERK) and ROS. In addition, the pro-differentiation factor Id-2 is upregulated by CBD. The anti-metastatic activity was also confirmed in animal models, in which CBD significantly reduced the primary tumor mass as well as the size and number of metastases in the lung (150). The study by Elbaz et al. showed that CBD inhibits the growth and metastasis of breast cancer cells by inhibiting epidermal growth factor (EGF)/EGFR signaling and modulating the tumor microenvironment, and that it can significantly inhibit EGF-induced proliferation and chemotaxis of breast cancer cells. Inhibition of EGF-induced activation of the EGFR, ERK, protein kinase B (AKT) and nuclear factor kappa B (NF-κB) signaling pathways and the secretion of the matrix metallopeptidases (MMP2 and MMP9) has been described. In in vivo models, the analysis of the molecular mechanism showed that CBD significantly inhibits the recruitment of TAMs in the primary tumor stroma and in secondary lung metastases (151). The selective CB2R agonist JWH-015 reduced the primary tumor burden and metastasis of the luciferase-labeled murine TNBC 4T1 cell line in immunocompetent mice in vivo and reduced the viability of murine 4T1 cells in vitro by inducing apoptosis. The reduction in cell viability mediated by JWH-015 was not dependent on Gαi signaling in vitro, nor was it altered by classical pharmacological blockade of other CBRs (CB1R, GPR55, TRPV1). The effect of JWH-015 was calcium-dependent and led to changes in mitogen-activated protein kinase (MAPK)/ERK signaling (152). Rimonabant (SR141716), a CB1R antagonist, inhibits the proliferation of the TNBC cell line MDA-MB-231 more effectively than ER+ cell lines. It also shows an antiproliferative effect in vivo by reducing the volume of xenograft tumors induced by injection of MDA-MB-231 in mice. Rimonabant inhibits the growth of human TNBC cells via a CB1R-lipid raft/caveolae-mediated mechanism (153). A strong synergy between rimonabant and erastin in inhibiting the growth of TNBC cells has been described both in vitro and in vivo. This occurred by increasing the levels of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and ROS production in the cytosol, enhancing intracellular glutathione (GSH) depletion and inducing G₁ cell cycle arrest (154). Cannabidiolic acid activates the expression of PPARβ/δ target genes in the MDA-MB-231 cell line (155). The molecular interaction between the cannabinoid agonists of vetiver oil and the CBR2 receptor was found to be the cause of the cytotoxicity of vetiver oil on the 4T1 cell line (156). Styrene-maleic acid nanomicelles encapsulating WIN55,212-2 were synthesized to reduce the side effects and increase the efficacy of the drug. The synthetic cannabinoid analog WIN55,212-2 is a synthetic CBR agonist with a cytotoxic effect on the MDA-MB-231 cell line, but causes psychoactive side effects (157). The synthesis and evaluation of a selective, non-psychotropic CB2R agonist, designated compound 10, with in vivo activity against MDA-MB-231 cells was reported. This novel cannabinoid o-quinone (compound 10) has been described to induce cell apoptosis through CB2R activation and oxidative stress. Importantly, compound 10 showed no toxic effects on non-cancerous human mammary epithelial cells or in vivo (158). Photodynamic therapy for TNBC was combined with CB2R agonists for TNBC. Synergistic effects were observed in both in vitro (MDA-MB-231 cell line) and in vivo experiments. The survival time of tumor-bearing animals was significantly prolonged (159).

7. Cannabinoids and triple-negative breast cancer treatment

8. Discussion

Cannabinoids are already being used in palliative care for cancer patients (182). However, the panel of the Multinational Association of Supportive Care in Cancer (MASCC) advises against the use of cannabinoids as an adjuvant analgesic for cancer pain (183). In addition to breast cancer, cannabinoids influence tumor progression in various types of cancer, e.g. glioma/glioblastoma, skin, liver, colon, prostate (184–186). TNBC TME could be influenced by cannabinoids, however the role of CBRs on different cells infiltrating the TME is largely unknown (187). Preclinical and clinical data show that cannabinoids can help with chemotherapy-induced neuropathy (167–170). In addition, there is a worldwide trend toward legalization (e.g., in the United States and Canada) of recreational and medical use of cannabis and cannabis products, with new health concerns arising related to the increasing use of cannabis (188). Exposure to cannabis and the cannabinoids (THC, CBD and CBG) may be a risk factor for the development of breast cancer in the female population (189, 190). Therefore, many breast cancer patients are exposed to cannabinoids in one way or another during and/or after their oncologic treatment, but the safety of this exposure is uncertain and requires further research. Many patients take cannabinoids in the belief that this will help cure their disease, although there is currently no clinical data to support this claim in breast cancer patients, including TNBC. A survey of breast cancer patients found that 42% of survey participants used cannabis to treat symptoms and about half of these participants believed that cannabis could treat the cancer itself. Most participants used cannabis during active cancer treatment (191). There are no clinical data on whether cannabinoids interfere with standard TNBC treatment, although initial clinical trials show that patients with metastatic non-breast cancer who use cannabis while receiving ICI immunotherapy have poorer outcomes. The ability of cannabinoids to shorten the survival of patients treated with immunotherapy suggests the efficacy of cannabinoids and the potentially important role of the endocannabinoid system in generating the immune response triggered by ICI (177, 178). At the preclinical level, there is increasing evidence that the endocannabinoid system may play a role in tumor growth, treatment resistance, and metastasis. In most preclinical studies, various cannabinoids inhibited breast cancer cell lines and improved cancer outcomes in TNBC animal models (148–150, 192–194) usually without interfering with standard treatment (e.g., cisplatin, epirubicin, doxorubicin, paclitaxel) (162–165). However, tumor-promoting effects of cannabinoids have also been reported. An antagonistic effect of CBD on TNBC cell lines treated with cisplatin at intermediate doses was observed (166). THC exposure has been shown to stimulate the growth of the ER+ MCF-7 cell line (195) and can lead to increased growth and metastasis of tumors with little or no expression of cannabinoid receptors due to suppression of the antitumor immune response (176). Importantly, cannabinoids have immunosuppressive properties (39–43). Immunosuppression in the periphery occurs mainly through the modulation of CB2R, while CB1R are less involved in this process (44–46). Due to the current limitations of immunotherapy (non-response, disease progression and hyperprogression), new strategies are needed to sensitize cancer cells to immunotherapy (196) and modulators of CBRs may provide a new mechanism to achieve this goal (Figure 1). CBRs could be a predictive biomarker for ICI therapy or a novel mechanism to reverse resistance to ICI therapy. Importantly, phytocannabinoids are not selective for CBRs and therefore give us little insight into the role of CBRs in the pathophysiology of cancer. Selective CB2R agonists and antagonists are needed to develop potential anti-cancer drugs that target the endocannabinoid system (197).

Author contributions

LD: Writing – original draft, Visualization, Validation, Supervision, Investigation, Conceptualization. SB: Writing – review & editing, Validation. ND: Writing – review & editing, Validation. SKB: Writing – review & editing, Visualization, Validation, Supervision, Conceptualization.

Funding Statement

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The research was supported by the Slovenian Research and Innovation Agency (ARIS) (grant numbers J3-3062 and P3-0003).

References