Although the EMT is both highly conserved and vital for normal developmental processes [
78], it serves an essential role in metastasizing cancer cells [
79]. In cancer pathogenesis, EMT promotes the dissemination of the primary tumor [
80]. EMT transcription factors (TFs), such as TWIST1, SNAIL1, and SLUG, are contributory to BC metastatic potential and associated with poor prognosis [
81]. ADAM12, a long splice variant with a transmembrane domain and member of the disintegrin and metalloproteinase family [
82], can be induced by Twist1, thereby promoting tumor invasion via regulation of invadopodia formation and focal adhesions [
83]. MiR-34a suppresses BC metastasis by downregulating EMT-TFs (SLUG, TWIST1, and ZEB1/2) and NOTCH1 signaling [
81]. Further, ADAM12 is a direct target of the miR-29 and miR-200 families, both involved in BC progression [
54]. Aside from regulating EMT-TFs, miRNAs can also regulate cytoskeletal rearrangement in cancer cells by targeting the expression of key molecules and cell signaling pathways involved in cell adhesion [
84]. MiR-8084, miR-708-3p, miR-96-182-183 cluster, miR-484, miR-210, and miR-142-3p modulate the invasive potential of BC cells by modulating EMT [
16‐
19]. Recently, it has been shown that miR-124, miR-199a/214, miR-3178, miR-30a, miR-508-3p and miR-212-5p can modulate the level of EMT markers and TFs regulating the expression of E-cadherin in TNBC, a subtype that commonly metastasizes to the brain [
51‐
53,
85].
Once breast tumor cells change their phenotype through EMT-driven mechanisms, metastasizing tumor cells start the process of metastasis by intravasation into nearby capillaries to facilitate neovascularization for survival [
86]. To metastasize at distal sites, cancer cells begin contacting endothelial cells via adhesion molecules and protein receptors [
86]. They then follow an amoeboid motility pattern and squeeze themselves between endothelial cells [
87]. Some secretory miRNA can regulate the integrity of the endothelium, and thereby the process of intravasation. For instance, miR-105 that is secreted by BC cells disrupts the endothelium by targeting Zonula occludens protein-1(ZO-1), a tight junction protein1 (TJP-1) [
29], thus promoting BM. Deryugina et al. discovered an alternative intravasation model suggestive of intravasation within the interior core of a primary tumor in parallel to stromal invasion [
88].
Angiogenic factors and growth factors either released by tumor cells or stromal cells individually or during their mutual crosstalk contribute to intravasation [
89]. These factors allow tumor cells to invade through the basement membrane, adhere to the endothelial membrane, and pass through endothelial gap junctions to disseminate into the circulation [
86]. Although no miRNA has been reported to influence intravasation directly, they can regulate angiogenic signals by targeting angiogenic factors and protein kinases. A recent study discovered a novel role of TGF-β by tumor-associated fibroblasts (TAFs) in the organization of tumor blood capillaries. TAFs enhanced vessel coverage by pericytes, which are vascular cells that support capillaries [
90]. In this regard, mRNA profiling of miR-520/373 overexpressing metastatic MDA-MB-231 cells elicited a strong downregulation of TGF-β signaling. It has also been reported that miR-520/373 are instrumental in reducing metastasis through downregulating TGF-β dependent potent angiogenic factors such as plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and angiopoietin-like 4 (ANGPTL4) [
25]. In a separate study, miR-204 was shown to suppress vascularization and angiogenesis
in vitro and
in vivo through targeting pro-angiogenic ANGPT1 and TGFβR2 in BC [
24]. The miR-200 family could also play a role in regulating angiogenesis by directly targeting the pro-angiogenic cytokines IL-8 and CXCL1 in endothelial cells [
27].
When a primary tumor grows, circulating tumor cells (CTCs) are shed and enter the circulation. Most CTCs are phagocytosed or undergo apoptosis, leaving behind only a few surviving CTCs to arrive at the targeted organ. Metastatic tumors, as well as CTCs from the primary tumor, may exhibit characteristics different from those of their cell of origin. In order to survive, CTCs must overcome anoikis and immune surveillance once they detach from the primary tumor. One of the tools exploited by CTCs after entering the circulation is platelet activation; by inducing platelet aggregation, tumor cells are protected from immune surveillance, undergo cell arrest within the vasculature, and experience enhanced survival [
91,
92]. The CSCs phenotype of BC cells is associated with brain tropism in TNBC patients [
93‐
95]. Debeb et al. have shown that overexpression of miR-141 in the MDA-MB-231 cell line enhances its brain tropism in a tail vein injection mouse model. Further, knockdown of miR-141 inhibited the metastatic ability of inflammatory BC to the brain, suggesting that miR-141 protects cells in the circulation and helps with colonization in the brain [
31].
Platelets also contribute to immune evasion by CTCs from scavenging natural killer (NK) cells by enshrouding CTCs and releasing TGFβ and platelet-derived growth factor (PDGF) that directly inhibit the activity of NK cells [
96]. Platelet-derived microparticles (PMPs) are major repositories for miRs, and platelets can transfer miRNA contents and modulate gene expression in CTCs [
33]. PMP encapsulated miR-183 can suppress NK cell activation, possibly via the silencing of DAP12 a key accessory protein critical for surface NK receptor stabilization and downstream signal transduction [
32]. Platelets also contribute to attenuate the early formulation of a metastatic niche [
97]. Thus, platelet-derived miRNA also helps in the survival of CTCs after intravasation. The role of miRNA released by CTCs and the intravascular microenvironment in establishing a brain pre-metastatic niche formation warrants further investigation.
Once CTCs are able to survive in circulation, BC cells arrest in blood capillaries and start the process of extravasation, a process coordinated by many oncogenes [
98]. Many pairs of ligand-receptor molecules contribute to the process of extravasation, including selectins, integrins, cadherins, CD44, and immunoglobulin superfamily receptors [
99,
100].
Extravasation is a rate-limiting step for BCBM, as cancer cells must overcome the initial defenses imposed by astrocytes and other protective factors in the brain microenvironment [
101]. Astrocytes that are mobilized to the metastatic brain lesion at a very early stage of colonization induce apoptosis through the FasL-mediated pathway [
102]. In recent studies, several miRNAs have been described to target various members of the Fas-mediated apoptotic pathway. For example, miR-7, let-7c, and miR-21 regulate the expression of FasL [
55], while miR-200c regulates the induction of apoptosis through CD95 by targeting FAP-1 [
56]. Cancer cells release protease inhibitors known as serpins to combat the apoptotic effects exerted by astrocytes. MiR-21 has been shown to inhibit Serpin1, a gene with novel tumor-suppressive effects in gastric cancer [
103]. However, its role in BM is unknown. Eventually, astrocytes support CTCs survival in brain parenchyma via establishing connexins (Cx) gap junctions and promote BM [
104]. The expression of miR-206 is inversely correlated with Cx43 levels and is associated with decreased proliferation and migration [
57]. PCDH7 in brain tropic BCs contributes to establishing Cx43 gap junctions with astrocytes and forms Ca
++ channels [
104]. A high PCDH7 level in the brain tropic CSC population has been reported and contributes to CSC extravasation, adaptation, and colonization in the new niche formation through the PCDH7-PLCb-Ca2þ-CaMKII/S100A4 pathway involving PCDH7-mediated tumor–astrocyte interaction [
95]. In addition, miR-19a, miR-32, miR-124a, miR-130b, miR-148a, and miR-583 have been reported as potential regulators of PCDH7 [
58]. However, the role of these miRNAs in PCDH7 regulated BM has yet to be studied [
95].
The production of IL6 and IL-8 by cancer cells requires the establishment of gap junctions with astrocytes [
105]. These cytokines influence both cell types by inducing the expression of endothelin ligand (ET-1) on astrocytes and endothelin receptors (ETAR and ETBR) on cancer cells [
101,
105]. ET-1 is regulated through miR-125a/b-5p in endothelial cells [
59]. In addition, the expression of a few genes was found to be dependent on such interaction [
106]. Some of them were validated in BM, such as TWIST1, GSTA5, and BCL2L1 [
106]. Interestingly, BCL2L1 is regulated by miR-1266, miR-185, and miR-30c [
60] in prostate cancer. TWIST1 is regulated by miR-151-3p in BC [
61]. These miRNAs are involved in negative regulation of the apoptotic pathway and upregulation of invasion or migration respectively, but the role in BM is not clear yet.
Emerging evidence shows that cell adhesion molecules (CAMs) play an essential role in extravasation through a cell-cell adhesion receptor. In an
in vivo model of BM, a subset of adhesion molecules, including E-selectin, VCAM-1, ALCAM, ICAM-1, VLA-4, and a4 were found to be upregulated in the cerebral endothelium when injected intracardially. Conversely, the expression of their ligands (PSGL-1, VLA-4, ALCAM, LFA-1, and VCAM-1) was upregulated in brain tropic cancer cells [
107], revealing a crucial role for these CAMs during the initial steps of extravasation. MiRNAs post-transcriptionally regulate CAMs. For instance, TGF-β induced ICAM-1, and E-selectin expression is regulated by miR-17 [
62]. MiR-126 and miR-1185 regulate endothelial expression of VCAM1 [
63,
108]. ALCAM is reported as a target gene of miR-483-5p [
64].
Moreover, cancer cells can invade through the endothelium by projecting invadopodia [
109]. Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis [
110]. PAK1 (P21 (RAC1) Activated Kinase 1) is responsible for guiding cancer cell extravasation in BCBM [
110]. PAK1 reduces the expression of miR-132 through the PAK1/ATF2/miR-132 axis. L1CAM, an adhesion molecule, mediates the spread of metastatic cells on the vasculature and additionally mediates interactions between cancer cells and endothelial cells in BM. The depletion of L1CAM in cancer cells fails to co-opt brain capillaries and hence is unsuccessful in promoting metastatic outgrowth. Interestingly, miR-21-3p was reported to be a positive regulator of L1CAM expression [
34]. These studies strongly suggest that miRNAs can modulate the expression of various CAMs in cancer, as well as endothelial cells, and thereby play a decisive role in the establishment of metastasis at the distant metastatic site via extravasation.
Reactive astrocytes have been shown to contribute to the formation of a protumorigenic niche via a number of mechanisms involving secreted molecules. In the BCBM mouse model, Massague’s group has identified 17 genes that are specifically correlated with BC-metastasis associated genes. Among these 17 genes, four genes, COX2, EGFR ligand HBEGF, ANGPTL4, and the a2,6-sialyltransferase ST6GALNAC5 were identified as signature molecules of BC metastasis to the brain parenchyma [
67]. COX2 is actively involved in BM by regulating the expression of MMP-1 in BC patients, and high expression is reported in BC patients [
111]. Interestingly, COX2 expression is associated with BBB permeability . COX2 induces a stem-like cell phenotype by upregulating miR-655 and miR-526b in BC, thereby rendering cells more metastatic [
66,
112]. MiR-212 directly targets HBEGF and suppresses cell growth, migration, and invasion [
65]. ST6GALNAC5, a direct target of miR-200c, is a specific mediator of BCBM [
67]. Conversely, the upregulation of ST6GALNAC5 in brain-tropic BC cells showed a decrease in adhesive properties of the endothelial component of a well-characterized human BBB
in vitro model [
113]. ST6GALNAC5 can also regulate the EMT process in BM and is a target of miR-200b [
68]. Several target genes actively participate in extravasation within the brain parenchyma, although their regulation in context of miRNA is not studied in BCBM.