Published OnlineFirst May 19, 2020; DOI: 10.1158/1541-7786.MCR-19-1229

MOLECULAR CANCER RESEARCH | REVIEW

Key Regulatory miRNAs and their Interplay with Mechanosensing and Mechanotransduction Signaling Pathways in Breast Cancer Progression Hamid Najminejad1, Behrouz Farhadihosseinabadi2,3, Mehran Dabaghian4, Asiyeh Dezhkam5, Esmat Rigi Yousofabadi6, Reza Najminejad7, Meghdad Abdollahpour-Alitappeh8, Mohammad Hossein Karimi9, Nader Bagheri10, Motahareh Mahi-Birjand11, Nasrin Ghasemi12, Mahta Mazaheri1, Seyed Mehdi Kalantar13, Alexander Seifalian14, and Mohammad Hasan Sheikhha15

ABSTRACT ◥ According to the WHO, breast cancer is the most common of miRNAs has been well-defined in the cancer process through cancer in women worldwide. Identification of underlying mechan- advances in molecular-based approaches. miRNAs are small isms in breast cancer progression is the main concerns of groups of RNAs (22 nucleotides) that contribute to various researches. The mechanical forces within the tumor microenvi- biological events in cells. The central role of miRNAs in the ronment, in addition to biochemical stimuli such as different regulation of various mediators involved in the mechanotransduc- growth factors and cytokines, activate signaling cascades, resulting tion signaling has been well clarified over the last decade. Unbal- in various changes in cancer cell physiology. Cancer cell prolifer- anced expression of miRNAs is associated with different patho- ation, invasiveness, migration, and, even, resistance to cancer logic conditions. Overexpression and downregulation of certain therapeutic agents are changed due to activation of mechano- miRNAs were found to be along with dysregulation of mechan- transduction signaling. The mechanotransduction signaling is otransduction signaling effectors. This study aimed to critically frequently dysregulated in breast cancer, indicating its important review the role of miRNAs in the regulation of mediators involved role in cancer cell features. So far, a variety of experimental in the mechanosensing pathways and clarify how the cross-talk investigations have been conducted to determine the main reg- between miRNAs and their targets affect the cell behavior and ulators of the mechanotransduction signaling. Currently, the role physiology of breast cancer cells.

Introduction stromal cells, and tissue-specific cells. ECM, as a main niche for normal Breast cancer remains one of the most prevalent cancers among and tumor cells, plays an important role in cell hemostasis (8). women in the world. According to the WHO report in 2018, 627,000 Collagens are a major component of ECM. However, other compart- deaths have been reported from breast cancer, accounting for approx- ments, including hyaluronan and proteoglycans, participate in ECM imately 15% of all cancer-related deaths among women (1). The formation (9, 10). It is well-documented that the physical properties of etiology of breast cancer is a complex phenomenon in which many ECM, including stiffness and topologic features, profoundly affect genetic and epigenetic factors are involved (2, 3). As a tumor grows, its cancer stem cell behaviors (11). In the breast tumor microenviron- genetic and epigenetic features alter as a result of microenvironment ment, overexpression of various ECM components increases the changes and, even, therapeutic pressure. Moreover, changes in the stiffness of tumor niche, which alter the biological behaviors of cancer genetic and epigenetic features of cancer cells mutually have an impact cells (12). Breast cancer cells cultured on a stiff substrate showed an on the cancer microenvironment (4–7). The cancer microenviron- increased expression of breast cancer stem cell markers through ment is composed of many players such as extracellular matrix (ECM), activation of the ILK/PI3K/Akt pathway (12). It is also reported that

1Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi Uni- Sciences, Yazd, Iran. 13Research and Clinical Center for Infertility, Shahid versity of Medical Sciences, Yazd, Iran. 2Hematopoietic Stem Cell Research Sadoughi University of Medical Sciences, Yazd, Iran. 14Nanotechnology & Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3Neuro- Regenerative Medicine Commercialization Centre (Ltd), The London BioScience science Research Center (NRC), Iran University of Medical Sciences, Tehran, Iran. Innovation Centre, London, United Kingdom. 15Genetics and Biotechnology Lab, 4Research and Development Department, Razi Vaccine and serum Research Research and Clinical Center for Infertility, Shahid Sadoughi University of Institute, Agricultural Research Education and Extension Organization (AREEO), Medical Sciences, Yazd, Iran. Karaj, Iran. 5Department of Midwifery, School of Nursing and Midwifery, Iran- Corresponding Authors: Alexander Seifalian, The London BioScience Innova- shahr University of Medical Sciences, Iranshahr, Iran. 6Department of Genetics, tion Centre, 2 Royal College Street, London NW1 0NH, United Kingdom. Iranshahr University of Medical Sciences, Iranshahr, Iran. 7Department of Internal Phone: 4420-7691-1122; E-mail: [email protected]; and Mohammad Hasan Medicine, Faculty of Medicine, Rafsanjan University of Medical Sciences, Raf- Sheikhha, Shahid Sadoughi University of Medical Sciences, No. 2 Bouali Ave, sanjan, Iran. 8Cellular and Molecular Biology Research Center, Larestan Univer- Safayeh, Yazd 8916877391, Iran. Phone: 9835-3824-7085; Fax: 9835-3824-7087; sity of Medical Sciences, Larestan, Iran. 9Transplant Research Center, Shiraz E-mail: [email protected] University of Medical Sciences, Shiraz, Iran. 10Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Mol Cancer Res 2020;18:1113–28 Sciences, Shahrekord, Iran. 11Infectious Disease Research Center, Birjand Uni- doi: 10.1158/1541-7786.MCR-19-1229 versity of Medical Sciences, Birjand, Iran. 12Abortion Research Centre, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical 2020 American Association for Cancer Research.

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the adhesion of breast cancer stem cells to ECM components, such as Interestingly, there is a cross-talk between mechanotransduction hyaluronic acid through CD44, increases the expression of genes and cytokine-induced signaling pathways, leading to modulation of involved in cancer stem cell development and drug resistance, includ- cell behaviors within a tissue (36). Wendt and colleagues reported that ing NANOG/SOX2 and Multi Drug Resistance 1, respectively (13). treatment of MDA-MB-231 breast cancer cells with TGFb significantly Topologic features of ECM also alter the migration of cancer cells so increases the expression of the proline-rich tyrosine kinase 2 (Pyk2) that breast cancer cell invasion is facilitated when the collagen fibers gene (37). Pyk2, as a member of the FAK family, participates in actin radially align relative to tumors (14). These data showed that ECM reorganization (38). Wendt and colleagues showed that TGFb pro- must be considered as an important environmental factor that can motes the expression of Pyk2 through activation of Src and Smad4 affect the biological behaviors of breast cancer cells (Fig. 1.) In addition signaling pathways, leading to epithelial-to-mesenchymal transition to ECM, tumor cells encounter with different mechanical forces that (EMT)-mediated cancer cell invasion (37). It is a clear example of how alter their biological behavior. In recent years, various studies have interactions between mechanical and chemical signals can increase the been carried out to find the effect of different mechanical stresses on invasion of cancer cells. Changes in proliferation and progression of the various characteristics of cancer cells such as proliferation, inva- cancer cells can also occur due to interaction between mechanotrans- siveness, and metastasis (15–17). It is well documented that the duction mediators and oncogenes (39). Qin and colleagues showed mechanical signals possess a pivotal role in the cancer progression, that MDA-MB-231 breast cancer cells under low shear stress exhibit an in which many oncogene signaling pathways interact with those increased cell proliferation through activation of the MAPK/ERK/YAP activated by mechanical forces (18). Therefore, the cytoskeleton signaling (39). contractility of tumor cells by activation of mechanotransduction Angiogenesis is a critical stage in cancer progression. In the cancer pathways in response to the physical stimuli profoundly affect cell microenvironment, vessels have a nonconventional morphology and attachment, cellular shape, cell proliferation, and migration through arrangement with more permeability than normal vasculature (40). various players such as miRNAs (18). There are various reasons that justify this phenomenon. As the tumor miRNAs are a part of the noncoding RNA family, which play a key size increases, the need for the entrance of nutrients is responded by regulatory role in gene expression (19, 20). Currently, the relationship creating new vessels within the tumor microenvironment (41). between dysregulation of miRNAs and different pathogenic condi- Because of various mechanical forces in the tumor area, endothelial tions, especially cancer, has been well established so that the evaluation cells acquire an unusual structure that forms vascular with specific of miRNA expression has been introduced as a promising approach properties such as the high opening in the wall and loose cell–cell for cancer detection (21–24). The miRInform Pancreas test is a junction (41). In general, mechanical forces in the tumor microenvi- well-known example of using miRNAs as a biomarker for cancer ronment can be categorized into two major groups, including solid and diagnosis (25). With advances in molecular techniques, the role of fluid stresses (42). In the solid stresses, mechanical forces stem from a miRNAs in the regulation of a wide range of genes has been well combination of various components of the tumor microenvironment, documented (26–28). The interaction between the mechanotrans- including ECM, cancer cells, and tissue cells (42). As a tumor pro- duction signaling and miRNA expression has been an interesting gresses, the composition and concentration of ECM alter and differ topic for researchers to find the underlying mechanisms involved in from the ECM contents in the normal tissue (43). It is well documented cancer cell behaviors in their microenvironment. The aim of this that the amount and stiffness of ECM in tumors are remarkably higher study was to review the literature to clarify the interaction between than that in normal tissue. The composition and concentration of miRNA dysregulation and the mechanotransduction signaling in ECM alter during tumor progression, which directly influences the breast cancer cells. formation of the capillary network by endothelial cells (44–46). It has been reported that endothelial cells on a highly cross-linked stiff matrix Mechanical Forces in Cancer exhibit higher cell growth, new vessel branching, and invasiveness Progression and Angiogenesis compared with those cultured on viscoelastic matrix (47). Treatment of tumors with b-aminopropionitrile, which reduces the stiffness of the The role of mechanotransduction in various biological events, such tumor, could remarkably decrease angiogenesis and new vessel as proliferation and migration, has been well investigated (29–31). In branching within the tumor microenvironment. In addition to the response to the ECM rigidity and stiffness, the focal adhesion (FA) ECM, a continuing increase in the number of cancer and stromal cells complexes and junction-related proteins trigger a series of inter- within the tumor microenvironment causes excessive mechanical actions that eventually convert extracellular mechanical forces into stress to the endothelial cells, which alters their morphology and gene an intracellular response (32). Mechanical forces can also directly expression (48). activate the ion channels and induce actomyosin contractility (33). In the fluid stresses, transmitted mechanical forces into the newly Integrins are oligomerized following the mechanical signals gener- formed vessels are produced by the flow of blood into the tumor ated by the interaction between cells and ECM. The oligomerized microenvironment. The presence of leaky vasculature, as well as integrins are then activated and form a complex with talin proteins, ineffective intratumoral lymph vessels, remarkably increases the inter- leading to increased intermolecular interactions (34). The focal stitial fluid pressure, so that its amount has been reported about 4 to complexes are formed following the initial interaction between 60 mm Hg in various solid tumors (49, 50). Flow stresses, especially integrins and adhesion plaque proteins. Eventually, the focal com- shear flow and basal-to-apical stresses, have an important role in plexes are assembled and matured into the FA structures (34). activating signaling cascades in endothelial cells, which changes their Taking together, interaction between integrins, actomyosin fibers, morphology, resulting in the alteration of vascular networks (51, 52). It and signaling mediators increases the proliferation and invasion of is reported that the transmission of basal-to-apical flow stress to cancer cells. (Fig. 2.) In addition to the FA structures, the presence endothelial cells gives an invasive character to these cells through of adherens junctions plays a critical role in the transmission of activating the FA kinase pathway as well as alteration in the cell–cell intercellular mechanical signals through various mediators such as junctions (53). As a result of such stresses, tumor endothelial cells cadherins and catenins (35). exhibit a range of distinct characteristics, including constant activation

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Figure 1. ECM overexpression within the tumor microenviron- ment occurs as a result of imbalance between ECM synthesis and production of ECM-remodeling enzymes. Moreover, the ECM composition of tumor environment differs from normal tissue due toa higher cross-linking rate, posttranslational modifications, and changes in the proportion of matrix compartments. Changes in ECM physical properties activate various mechano- Mechanical transduction cell signaling pathways, eventually alter- forces within ing cell behaviors including cell proliferation, cell inva- breast tumor siveness, cell drug resistance as well as angiogenesis.

ECM Cell populations overexpression and fluid occurs within ECM pressure increase the tumor site composition within the tumor and topographic site features change in tumor site

Activation of mechanotransduction signaling pathways

Changes in Cell invasion Cell proliferation Drug resistance Angiogenesis of survival, angiogenic, and activating signaling pathways (54). These It was reported that the import of transcriptional factors into the findings highlighted the importance of physical forces in tissue nucleus is controlled during mechanical pressure to the cell and homeostasis so that abnormal changes in mechanical forces within nucleus (ref. 57; Fig. 3.) For example, the entrance of myocardin- cell microenvironment alter their characteristics and may lead them to related transcription factors (MRTF) into the nucleus is dependent on form tumors. G-actin concentration and cell polarization. This transcription cofac- tor is inactive when binding to G-actin in the cytoplasm. As cell Mechanotransduction and Gene polarization increases, the actin polymerization into the F-actin fibers is elevated, resulting in dissociation of MRTF and G-actin (57). Upon it Expression release, MRTF enters the nucleus and binds to the serum response The presence of linker structures, including the LINC complex, on factor, leading to the expression of genes involved in the regulation of the nuclear envelope enables the physical connection between the actin dynamics. Moreover, following diminished polymerization of nucleus and cytoskeleton (55). Furthermore, the intranuclear network actin fibers, the import of the p65 transcription factor into the nucleus of structural proteins, known as nuclear lamina, facilitates the trans- increases, while MRTF is exported to the cytoplasm (57). It was shown duction of mechanical forces into the nucleus, resulting in a sense of that the mechanical forces could alter the import and export of physical forces by the nucleus. It was reported that proteins forming transcriptional factors into the nucleus. nuclear lamina, mainly lamins, serve also as a mechanosensor, which It is suggested that the morphology and organization of the nucleus regulates the function of transcription factors (56). Despite the exten- have a great impact on gene expression. As mentioned before, there is a sive efforts to understand the way that the nucleus senses mechanical connection between the nucleus and cytoskeleton, so that any alter- forces, little is known regarding the mechanisms underlying how these ation in actomyosin structure changes the 3D organization of the mechanical signals induce changes in gene expression. However, nucleus (58). One clear example is the upregulation of dihydrofolate various studies suggested different mechanisms in this context, as reductase transgene through applying local forces to the cell sur- discussed below. face (59). In a study, Tajik and colleagues evaluated the effect of shear

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ECM

a b

Integrin

Talin Focal adhesion Paxillin complex a-Actin Kindlin Actin fibers Vinculin Arp2/3 complex

FAK

Nesprin

Nuclear lamina Emerin SUN1/2

Figure 2. The interaction between FA complex and cell nucleus. In the cytoplasmic side, the complex of Talin, Paxilin, and Vinculin forms an initial anchor to accumulate proteins involved in focal adhesion. The actin polymerization occurs at the focal adhesion complex site and, then, the polymerized fibers are stabilized and branched through proteins such as a-actin and Arp2/3 complex. Binding of actin filaments to a protein complex located in the nucleus membrane is responsible for the transmission of physical forces to the cell nucleus, leading to changes in the structure of nuclear lamina and gene expression.

stress applied by the three-dimensional magnetic twisting cyto- cis element and promoters of the cytokine-related genes. Moreover, metry technique on the expression of the GFP-tagged DHFR chromatin remodeling has a great impact on master regulation of a transgene. They reported that applying shear stress to cells results cluster of genes such as the Hox gene cluster (60). These changes in in chromatin stretching in the direction of applied stress that the chromatin structure have been also observed following physical upregulates the expression of DHFR. It was found that the amount stress to cells where the polarized fibroblasts exhibit a localized of upregulation of DHFR is proportional to the amount of chro- functional gene cluster within the intermingling regions of the matin stretching (59). chromatins (58). However, there appear to be other mechanisms The spatial arrangement of genes seems to be very critical in the for the effect of mechanical forces on gene expression. Future regulation of gene expression. Once cells are stimulated with studies in this area will help clarify the role of mechanical forces cytokines,theformationofchromatinloopsbringstogetherthe in the regulation of gene expression.

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Physical stress Transcription factors entrance Gene expression due to change in the cytoskeleton TF TF rearrangement

TF Nuclear TF lamina

Actin filaments

Nucleolus LINK complex

Chromatin

Physical stress may change the spatial arrangement of genes

Figure 3. The suggested mechanisms for how mechanical forces affect gene expression. Until now, some mechanisms have been suggested to describe the effect of physical forces on gene expression. It was found that mechanical forces increase the entrance of different transcriptional factors into the nucleus. Moreover, the interaction between LNIK complexes and cytoplasmic actin fibers transmits the mechanical forces to the nuclear lamina network, which alter the 3D organization of chromatins and gene expression. In addition, mechanical stresses may affect the spatial arrangement of genes, facilitating the juxtaposition of cis elements and gene promotors. TF, Transcription factor. miRNA Synthesis: Its Role in Breast modifications, such as capping, polyadenylation, and splicing, are introduced to the pri-miRNAs (66). In the next step, the stem-loop Cancer structure of pri-miRNA serves as an identifying mark for Drosha miRNAs, as a main member of noncoding RNAs, are found in a complex, resulting in the separation of a hairpin loop with 70 nt wide range of organisms (61). This type of regulatory molecules has an (pre-miRNA) by this enzyme (67). The Drosha microprocessor important role in gene regulation, whose unbalanced expression leads complex consists of Drosha, an RNA-binding protein called to different diseases, especially cancer (62). The first reports on the role DGCR8, and various cofactors (67). Downregulation of Drosha of miRNAs in breast cancer dates back to 2005. Since then, a variety of was observed in triple-negative breast cancer and patients with studies have been conducted each year to further clarify the role of breast cancer with the higher tumor volume and histologic grade. these molecules in breast cancer (63). Moreover, overexpression of DGCR8 was found in breast cancer þ þ RNA polymerase II, although responsible for miRNA transcription, samples with characteristics including Ki67 / ER as well as was demonstrated to play a role in transcription of a group of miRNAs invasive ductal breast carcinoma (68). has been discovered (64). miRNAs are encoded in different parts of the Following processing of pri-miRNAs to pre-miRNAs by the Drosha genome, including the intron of protein-coding genes as well as the microprocessor complex, pre-miRNAs are exported to the cytoplasm intron and exon of noncoding RNAs (65). miRNA synthesis is a through binding to a transporter complex composed of exportin-5 multistep process in which various enzymes are involved. At the first (XPO5) and Ran-GTP (69). Overexpression of the aforementioned step, miRNA precursors are transcribed by RNA polymerase, leading transporter complex is along with an invasive phenotype in breast to formation of a pri-miRNA with a stem-loop structure. Then, various cancer (70, 71).

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At the final step, pre-miRNA in the cytoplasm binds to the RISC decreased expression of miR-551a in tumor samples as compared complex, which is converted to a duplex miRNA with 22 nt with the normal tissue around the tumor site. In this light, forced length. This double-strand miRNA is separated and forms guide expression of miR-551a in MDA-MB-231 and ZR75 cancer cell lines and messenger strands (72). The interaction between Ago proteins led to decreased expression of FAK, which was dependent on miR- and guide strand miRNA forms a complex for recognition and 551a concentrations (86). Further analyses using miR-551a antago- cleavage of target mRNA. In addition to the direct role of miRNA in mirs exhibited an elevated expression of FAK in the aforementioned mRNA degradation, these molecules regulate the expression of their cell lines. In general, forced overexpression of miR-551a in the MDA- targets in the translational level (72). Changes in the expression of MB-231 cells led to a decrease in cell proliferation as well as colony- the RISC complex were frequently observed in breast cancer. For forming ability (86). An in vivo study demonstrated that nude mice example, the expression level of the Ago2 protein was significantly receiving miR-551a-transfected MDA-MB-231 exhibit lower tumor lower in highly invasive breast cancer as compared with nonneo- values as compared with those receiving empty vector–transfected plastic ones (73). Based on the above, any dysregulation in the cells. The miR-551a expression was reported to be mainly promoted by expression of mediators involved in miRNA processing influences c-Fos, so that the expression of this transcription factor, similar to miR- the synthesis of miRNAs, which disrupts the expression balance of 551a, is remarkably lower in the breast tumor samples as compared their target. Accordingly, an overwhelming majority of studies with breast normal tissues (86). confirmed the difference between miRNA patterns of breast cancer miR-7 is another miRNA involved in FAK regulation in breast samples and normal breast tissue, highlighting the importance of cancer cells. A study on the FAK protein level revealed a significant these regulator molecules in the balance of physiologic events in difference in various breast cancer cell lines (87). The FAK protein level normal cells (69, 71, 74–77). was found to be related directly to the degree of invasiveness of various According to the important effect of mechanical forces in the breast cancer cell lines. Results obtained from that study showed that progression of breast cancer, in the following, we discussed the role the amount of FAK in HBL-100, as a nonmalignant human mammary of miRNAs in the regulation of mechanotransduction mediators in epithelial cell, was lower than those in breast cancer cell lines with breast cancer (Table 1). Even though the mechanotransduction moderate invasiveness (MDA-MB-468, MDA-MB-453, and MCF-7), signaling is a complex subject, including the interaction between a while the highest amount of the FAK protein was observed in MDA- wide varieties of mediators, we attempted to separately review the MB-435s, BT-549, and MDA-MB-231 cells that exhibited a highly cross-interaction between miRNAs and different stages of mechan- invasive phenotype (87). However, the expression of miR-7 indicated otransduction pathways. an inverse pattern as compared with FAK, such that the noninvasive and moderately invasive cell lines showed a higher expression rate of miRNAs and Mechanotransduction miR-7 than highly invasive breast cancer cells. As a result of forced miR-7 overexpression in the MDA-MB-435s and MDA-MB-231 cells, Pathways EMT was significantly decreased (87). EMT is a phenomenon in which The role of miRNAs in the regulation of mediators involved in the morphologic changes in epithelial cells cause them to find a structure mechanotransduction signaling is important to understand how similar to mesenchymal cells, which is a vital precondition for various changes in mechanotransduction mediators through miRNAs affect developmental processes of cancer cells such as metastasis (88). proliferation, invasiveness, and migration of breast cancer cells (Fig. 4.) Transient overexpression of miR-7 in MDA-MB-435 and MDA- In the following sections, we discussed the role of miRNAs in the MB-231 cells changed their spindle fibroblast-like morphology to a regulation of different mechanotransduction mediators. typical epithelial cobblestone-like shape. Moreover, the transient overexpression of miR-7 in the mentioned cells led to decreased miRNAs and FA signaling expression of SNAIL, VIM, fibronectin and N-cadherin, and elevated FA structures are composed of the interaction between the actin expression of E-cadherin (87). Furthermore, overexpression of miR-7 cytoskeleton and transmembrane integrins that are in contact with the was accompanied by tumor growth and metastasis suppression in vivo. ECM components. Integrins with heterodimer units form a trans- However, the expression pattern of FAK and miR-7 is not the same in membrane receptor, mediating the first contact between the cytoskel- all breast cell lines (87). For example, T47D, which is categorized as a eton and ECM in the cell microenvironment (78). In the cytoplasmic breast cancer cell with moderate invasiveness, shows low expression of side, actin-binding proteins, such as talin, vinculin, and paxillin as well FAK and miR-7. This finding suggests that there may be other path- as other mediators including signaling effectors, such as GTPases, ways involved in the regulation of FAK and miR-7 in these cells (87). capping proteins, actin-binding proteins (ABP), phosphatases, phos- P130Cas is one of the important regulators in the mechanotrans- pholipases, and FAKs, enforce the interaction of integrins with actin duction signaling. This protein has interaction with various mediators cytoskeleton, resulting in further polymerization of actin fibers (79, 80). such as tyrosine kinases, guanine nucleotide exchange factors (GEF), FAK, a 125-kDa nonreceptor protein tyrosine kinase, is one of the key and other adaptor molecules. FAK and Scr proteins are the key elements in FA complexes. This protein is phosphorylated as a regulators of p130Cas by phosphorylation of this protein (89–91). downstream substrate for activated v-Src in response to integrin– p130Cas overexpression in breast cancer is accompanied by resistance ECM interaction (81, 82). As a tumor grows, FAK exhibits two main to tamoxifen and low survival rates (92, 93). miR-362-3p and miR-329 functions, including promoting tumor cell adhesion and providing an expression was found to be downregulated in breast cancer. The antiapoptosis signal (83). The correlation between overexpression/ P130Cas protein is the target for the abovementioned miRNAs, whose activation of FAK and tumorigenesis, invasion, and metastasis in expression increases in breast cancer as a result of downregulation of various cancer types has been described (84). Overexpression of FAK miR-362-3p and miR-329 (94). Forced expression of miRNAs 362-3p in the benign and invasive breast cancers is along with preinvasive and and 329 in MCF-7 cells exhibited that the expression rate of P130Cas aggressive phenotypes, respectively (85). Reports showed that there is significantly decreases in the transfected cells. DNA methylation is an inverse relationship between FAK and miR-551a expression in suggested as a possible mechanism responsible for downregulation breast cancer cells. The real-time expression analysis exhibited of the abovementioned miRNAs. It seems that MeCP2, as a CpG

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Table 1. miRNAs involved in the mechanotransduction cell signaling in breast cancer.

Expression status Target protein/ in breast cancer Target miRNA proteins cells Effect site Ref miR-551a/miR-7 FAK Downregulation Inhibitory effect on proliferation, invasion, and migration 30UTR 86, 87 miR-362-3p/ P130Cas Downregulation Inhibitory effect on proliferation, invasion, and migration 30UTR 94 miR-329 miR-10b Syndecan-1 Upregulation Induction of invasiveness and migration, induction of filopodium 30UTR 97 formation, down-regulation of E-cadherin miR-142-3p WASL/integrin-aV Downregulation Inhibitory effect on cell invasion, decreased cell size and membrane 30UTR 99 protrusion structures miR-30c TWF1/VIM Downregulation Decreased EMT, decreased FA formation, increased sensitivity to 30UTR 106 paclitaxel and doxorubicin miR-149 GIT1 Downregulation Inhibitory effect on cell invasion and migration, decreased FA 30UTR 111 formation miR-584 PHACTR1 Downregulation Inhibitory effect on cell invasion and migration, decreased FA 30UTR 119 formation miR-200c FHOD1/PPM1F Downregulation Inhibitory effect on cell invasion and migration 30UTR 128 miR-204 BDNF Loss of loci Decreased invasion, decreased lung metastasis in the mouse model 30UTR 140 miR-145 JAM-A/Fascin Downregulation Decreased cell invasion and filopodium formation, increased cortical 30UTR 143 actin distribution miR-490-3p RhoA Downregulation Inhibitory effect on cell invasion and proliferation, arresting cells in 30UTR 151 the G1 phase, decreased tumor growth in the mouse model miR-146a RhoA Downregulation Inhibitory effect on cell invasion and migration 30UTR 152 miR-135b LATS2 Upregulation Increased proliferation, migration, and colony formation 30UTR 166 miRNA-125a LIFR Upregulation Increased stem cell pool, increased non-malignant breast epithelial 30UTR 168 stem cells toward malignant cells miRNA-136-5p/ HERC4 Downregulation Inhibitory effect on the cell proliferation, survival, and migration, 30UTR 167 miRNA-1285-5p decreased tumor growth in the nude mouse model

Abbreviations: BDNF, brain-derived neurotrophic factor; FA, focal adhesion; FAK, focal adhesion kinase; FHOD1, formin homology domain protein 1; GIT1, G protein- coupled receptor kinase-interacting protein; JAM-A, junctional adhesion molecule-A; LATS2, large tumor suppressor kinase 2; LIFR: LIF receptor subunit alpha; PHACTR1, phosphatase and actin regulator 10; PPM1F, protein phosphatase Mg2þ/Mn2þ dependent 1F; RhoA, Ras homolog gene family member A; TWF1, twinfilin actin-binding protein 1; VIM, vimentin; and WASL, WASP like actin nucleation promoting factor. island–binding protein, plays a role in DNA methylation of miR-362-3p Matrigel invasion assay and confocal immunofluorescence microsco- and miR-329 promoters (94). py results, miR-142-3p–overexpressing cell lines were shown to have Syndecan-1 is a transmembrane proteoglycan that has a different remarkably lower invasiveness, protrusion formation, and cell size as role in cancer development, serving as a coreceptor for various growth compared with the control (99). factors and cytokines as well as modulating integrin function (95). The relationship between EMT and breast cancer chemoresis- Overexpression of miR-10b downregulates syndecan-1 expression in tance has been well confirmed in literature (102, 103). In the early breast tumor cells (96). Overexpression of this oncomiR led to stages of breast tumor formation, EMT plays an undeniable role in appearance of a preinvasive phenotype in breast cancer cell lines with resistance to endocrine therapy and chemotherapies (104). EMT is a nonmetastatic phenotype. Syndecan-1–depleted MDA-MB-231 cells regulated by various mediators in the cancer cells such as twinfilin 1 exhibited upregulation in genes involved in motility and invasiveness (TWF1), which is known as an actin-binding protein. This protein such as COX-2, actin g 2, vinculin, cadherin-11, MYL9, ATF-2, has two ADF-H domains that prevent the actin polymerization transgelin-1, and RhoA/C (97). Moreover, a tight adherence of syn- through interaction with the capping protein and sequestration of decan-1–depleted MDA-MB-231 cells to ECM components, including G-actins (105). miR-30c was found to be a key element in the fibronectin and laminin, resulted in further activation of FAK and downregulation of TWF1. Transient overexpression of this onco- RhoGTPases and an acquired invasive phenotype in these cells (97). miR in MDA-MB-231 cells not only decreased the F-actin forma- The formation of membrane protrusions is the critical step in cell tion and EMT, but also increased the sensitivity to paclitaxel and motility and FA complex formation (98). miR-142-3p decreases doxorubicin at low doses (106). Furthermore, vinculin staining membrane protrusions and invasiveness in various breast cancer cells showed a significant decrease in the FA formation in the transfected through direct targeting of 30 UTR of WASL and integrin-aV (99). cells. An attempt to find the transcription factor responsible for These two proteins actively participate in metastasis (100, 101). miR-30c expression led to introduction of GATA3, whose over- Schwickert and colleagues studied the role of miR-142-3p in the expression in the GATA3-deficient MDA-MB-231 increases the prevention of breast cancer cell invasiveness. They reported that forced expression level of miR-30c (106). overexpression of this oncomiR in various cell lines, including MCF-7, The GIT1 protein is one of the key regulators of FA formation and MDA-MB-468, and MDA-MB-231, effectively decreases the expres- cell migration (107, 108). This protein has a complex structure, sion level of WASL and integrin-aV (99). In addition, transfected cells containing various domains and effector sites (109). In the cell showed a lower expression rete of the genes involved in cytoskeleton migration phase, this protein is colocalized with FAK and paxillin motility and regulation such as RAC1 and CFL2. According to the within the FA points and interacts with a verity of mediators such as

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miRNAs and the cytoskeleton Cooperation of the actomyosin network and proteins involved in Physical features of breast tumor FA complexes create micromotor devices, which are a prerequisite for microenvironment the mechanotransduction signaling. The actin cytoskeleton network is an interconnected and complex structure which is composed of a dynamic interaction between different components, such as G-actin, F-actin, and actin-binding mediators (112). Formation of such struc- tures is a primary step in various biological events in tumor cells, especially metastasis. A deep understanding of the metastasis process is miRNA achievable by a detailed study on the mechanisms involved in cell Dysregulation motility and invasion in response to microenvironment stimuli. Cell movement controllers in normal cells severely constrain cell motility and migration within the tissue. In cancers, metastasis will occur as due to dysregulation of controller elements, such as miRNAs (113). Vimentin (VIM) is one of the main cytoskeleton structural proteins Changes in expression of : that participates in the formation of intermediate filaments (114). This Focal adhesion complex mediators Cell proliferation protein involves metastasis, invasion, as well as EMT in cancer Actin binding proteins cells (115). Results obtained from an in vitro study revealed that GTPase family members Cell invasiveness Mechanotransduction signaling Cell metastasis transfection of the vector containing miR-30c in the breast cancer effectors cell line (MDA-MB-231) directly targets the 30 UTR of VIM (105). As a result, downregulation of VIM led to a remarkable decrease in cell Figure 4. invasion of miR-30c–transfected MDA-MB-231 cells. The contribu- The effect of physical forces on the expression of mechanotransduction med- tion of VIM in cell invasion was further confirmed by knocking down iators. Breast tissue cells in the tumor microenvironment exhibit a dysregulated using siRNAs, where the VIM knocked down MDA-MB-231 showed a miRNA expression pattern, which affects the expression of various mediators in higher mesenchymal-to-epithelial transition (MET) phenotype com- the mechanotransduction signaling. Changes in the expression of mechano- transduction signaling effectors profoundly influence cell behaviors, including pared with the control (105). cell proliferation, invasiveness, and metastasis. The TGFb signaling has a double-edged sword role in breast cancer progression. TGFb has an antitumor function at the early stages, inhibiting tumor initiation and progression, while serving as a pro- metastatic agent at advanced stages (116, 117). Indirectly, TGFb GEFs, PAK complex, Rac1/Cdc42, and PIX, which consequently induces metastasis in breast cancer through upregulation of protein promote the protrusion structures (110). The examination of primary phosphatase and actin regulator 1 (PHACTR1), which is known as an breast tumors and, even, lymph node metastases revealed a higher actin-binding protein. PHACTR1 is a member of the actin-binding expression level of GIT1 compared with the surrounded normal protein family with RPEL repeats (118). This protein was found to tissues (111). Furthermore, the expression rate of miR-149 was found regulate the actin polymerization/depolymerization dynamic so that to be remarkably lower in the tumor samples. There is an inverse knocking down of PHACTR1 results in disorganization of actin relationship between miR-149 and GIT1 expression in breast tumor filaments. TGFb, via the smad signaling, decreases miR-584 expression cells. Increased expression of GIT1 and downregulation of miR-149 that directly targets 30UTR of PHACTR1 (119). Study on different have been reported in the advanced stages of breast cancer (111). breast cancer cell lines revealed that overexpression and downregula- 0 In vitro analysis showed that miR-149 directly targets the 3 UTR of tion of PHACTR1 and miR-584, respectively, are observed only in GIT1. Forced overexpression of miR-149 in IV2-1, IV2-2, and Hs578T invasive basal-like breast tumor cells including SUM159PT, MDA- led to the suppression of cell migration and invasion. As a result of MB-231, and SCP2. In the TGFb-stimulated SCP2 cells, forced over- GIT1 downregulation, the FAK-mediated downstream signaling are expression of miR-584 resulted in a decrease in PHACTR1 expression impaired due to decreased FAK autophosphorylation at Y397, FAK and TGFb-dependent cell migration (119). Confocal microscopic phosphorylation at Y861, and paxillin phosphorylation at Y118. analysis of the cells transfected by miR-584 exhibited an accumulation Moreover, GIT1 downregulation is along with increased lysosomal of stress fiber in these cells. Despite the formation of stress fibers in the degradation of a5b1 integrin complexes, which negatively influences transfected cells, these cells were unable to form filopodia, suggesting the FA complex formation (111). It explains how GIT1 downregula- that downregulation of PHACTR1 stops the actin treadmilling tion interferes with the FAK–Src signaling. However, it seems that cycle (119). decreased phosphorylation of paxillin is not only due to the inefficient The miR-200 family is essential for epithelial cells to represent an FAK–Src signaling. Paxillin instability also occurred as a result of epithelial phenotype. To occur EMT in most epithelial cells, expression increased proteasomal degradation as well as loss of contact with GIT1, of these oncomirs must be stopped, indicating the important role of which were observed in GIT1-downregulated IV2 cells. This highlights the miR-200 family in cancer development (120–122). The morpho- the important role of GIT1 as a stabilizer for a5b1 integrins and other logic changes in EMT force the immobile epithelial cells toward mediators such as paxillin, which profoundly affect the formation of mesenchymal characteristics with an intrinsic proinvasive proper- FA complexes (111). ty (123). Downregulation of these oncomirs has been confirmed in the Until now, various adaptor proteins have been identified to be metastatic stages of cancer development compared with early involved in the FA complex. Our knowledge regarding how FA one (124, 125). It is confirmed that lower expression rates of miR-200 complexes are formed is increasing. It is obvious that further inves- correlate with poor prognosis in various human epithelial malignan- tigations will be necessary to detect the unknown mediators and cies (121, 126, 127). FHOD1 and PPM1F proteins are a downstream miRNAs in this context. target of miR-200c. These proteins, as downstream mediators of the

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RhoA signaling, facilitate the formation of bundled actin stress 3, and MCF-7 cell lines revealed decrease expression of JAM-A, fascin, fibers (128, 129). FHOD1 promotes expression of myosin light podocalyxin, and Serpin E1, and increased expression of MYL9, chain (MLC2) through nuclear accumulation of MRTF-A, known as gamma-actin, and transgelin. Moreover, forced overexpression of this a SRF coactivator, (128, 130). On the other hand, PPM1F elevates the oncomiR in MDA-MB-231 cells remarkably reduced cell invasion and phosphorylation rate of MLC2 in an MLC2 expression–independent filopodia formation, but increased cortical actin distribution (143). manner. PPM1F was reported to increase phosphorylation of myosin In a study, in silico analysis was used to identify the cross-interaction light chain phosphatase (MYPT) through inhibition of PAK at its between various miRNAs and their targets by a network-based inhibitory site (131). However, Jurmeister and colleagues reported that approach; findings from that study demonstrated that miRNAs, miR-200c overexpression in MDA-MB-231 results in increased MYPT including miR-612, miR-940, and miR-661, are involved in cytoskel- phosphorylation at Thr696 or Thr853 sites, while the PPM1F silencing eton regulation of breast cancer cells (146). This claim was tested using had no impact on phosphorylation of any sites on this protein. Their the impact of these miRNAs on cytoskeleton changes in a RPE1 cell results showed a decrease in cell migration and invasion following model. Overexpression of miR-612 and miR-940 inhibited myosin II forced overexpression of miR-200c in the MDA-MB-231 cells (128). phosphorylation and consequently led to decreased cell invasion in Another study also confirmed the inhibitory effect of miR-200 in cell RPE1 cells. In contrary, cell invasion and myosin II phosphorylation invasion and migration. In that study, overexpression of miR-200 increased following forced overexpression of miR-661 in RPE1 (miR-200a and miR-200b) in breast cancer cells was along with the cells (146). change in actin rearrangement (132). In fact, miR-200 overexpression Cytoskeleton dynamics play a pivotal role in different aspects of cell changed stress fibers to cortical actin, resulting in decreased invasion biology. The regulation of mediators involved in cytoskeleton dynam- and cell migration. miR-200 also inhibited the invadopodia formation ics by miRNAs is important so that changes in the expression of and stabilized FA complexes. miR-200 was proposed to target acti- miRNAs affect the normal cytoskeleton dynamics of normal breast vators of the Rho family, including ARHGEF3 and NET1, and some cells. Further studies will increase our knowledge regarding the role of targets in downstream of the Rho signaling, such as ROCK2, MYH9, miRNAs in cytoskeleton rearrangement, representing a promising MYH10, MPRIP, MYPT1, and MYLK (132). window for future therapeutic strategies based on the recovery of Chromosome loss occurs frequently in various cancer types, result- cytoskeleton rearrangement in breast cancer cells. ing in loss of functions of some essential genes involved in cell physiology and regulation (133). Different studies confirmed the loss miRNAs and Rho-family GTPases of chromosomal loci containing miR-204 in a verity of cancer The Rho-GTPases family includes different small G proteins with types (134, 135). The role of miR-204 as a tumor growth/metastasis GTPases activity that has an active role in various biological events suppressor is mainly related to the suppression of genes involved in such as cell motility. This protein family consists of more than 50 tumorigenesis such as brain-derived neurotrophic factor (BDNF). members with some shared features, including binding to hydrolyze This protein is known as a nerve growth factor that activates tropo- guanine nucleotides, having a molecular weight ranging from 18 to myosin-related kinase B (TrkB; refs. 136, 137). In tumor cells, the 28 kDa, and possessing polyisoprenylation regions at their C- BDNF/TrkB signaling involves in different steps of tumorigenesis, terminal (147). Several studies showed that any abnormal alterations including metastasis, differentiation, and proliferation (138). Upre- in the activity of these mediators lead to pathologic conditions, gulation of BDNF/TrkB in breast cancer correlates with poor prog- especially cancers. Rho GTPases, as main regulators of cytoskeleton nosis (139). Loss of miR-204 loci in breast tumor cells promotes rearrangements, control cell morphology, growth, and adhesion, so overexpression of BDNF/TrkB and activation of the AKT/mTOR/ that overactivation of these proteins is observed in malignancies (148). Rac1 pathway that reorganize the actin network, leading to cancer cell Therefore, understanding of mechanisms involved in Rho GTPases migration and invasion. Study on a mouse model revealed that dysregulation in cancers would help us find possible therapeutic systemic injection of MDA-MB-231 cells in the animals severely weapons against cancers. caused lung metastasis, while those group receiving systemic admin- RhoA is a well-known member of Rho GTPases, activating through istration of miR-204 showed a remarkable decrease in tumor growth various stimuli, including extracellular signals as well as growth factors and metastasis (140). and hormones such as insulin, PDFG, and EGF. Its activation is along miR-145 was reported to be downregulated in both breast cancer with different morphological changes toward lamellipodia formation cell lines and clinical tumor samples compared with normal breast through polymerizing actin filaments and nascent focal complex tissue around the tumor site (63, 141, 142). In normal breast tissue, formation (149). In the cell adhesion and spreading stage, inhibition miR-145 is exclusively expressed in myoepithelial and basal cells of of RhoA-GTP and concurrent activation of Rac1 and Cdc42 lead to mammary ducts and lobules, while its expression is diminished or suppressed actomyosin contractility and increased actin-mediated suppressed during tumor progression. miR-145 exerts its antimeta- protrusions (150). So far, few miRNAs have been reported to possess static effect on normal cells through direct targeting of JAM-A and a regulatory effect on RhoA expression in breast cancer. For example, fascin proteins (143). JAM-A, as a membrane protein, plays an active miR-490-3p is found to directly target the 30UTR of RhoA mRNA. The role in cell–cell junction as well as cell motility regulation (144). expression level of miR-490-3p is significantly lower in breast tumor Indeed, the PDZ-domain protein mediates the interaction between samples as compared with surrounding normal tissue. Transient the cytoplasmic domain of JAM-A and actin cytoskeleton that influ- overexpression of miR-490-3p in McF-7 and T47D cells confirmed ences cytoskeletal rearrangements (145). Fascin contributes to cell that overexpression of this oncomiR decreases the migration and migration through transforming lamellipodial structures into filopo- invasion ability of these cells through downregulation of RhoA, dia by bundling actin filaments. Obviously, overexpression of this MMP-9, P70S6K, and Bcl-XL proteins (151). In addition to cell protein promotes cancer cell migration (143). The luciferase activation migration, miR-490-3p could reduce cell proliferation through arrest- 0 – assay revealed that miR-145 directly targets 3 UTR of JAM-A and ing the transfected cell in the G1 phase. Cell proliferation inhibitory fascin, leading to downregulation of these proteins. Transfection of the effects of miR-490-3p are attributed to downregulation of P70S6K vector containing miR-145 in MDA-MB-468, MDA-MB-231, SK-BR- protein that serves as a downstream mediator of the PI3K/AKT

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pathway. An in vivo study revealed that subcutaneous injection of mediators. In various cancer types, LATS2 expression is downregu- MCF-7 cells transfected by miR-490-3p mimic in mice causes forma- lated by different miRNAs (164, 165). In breast cancer, miR-135b is tion of smaller tumors with lower RhoA expression as compared with responsible for direct 30 UTR targeting of LATS2. Study on miR-135b the control (151). expression exhibited that this miRNA is remarkably upregulated in RhoA expression is also influenced by miR-146a through direct breast tumor samples and cell lines as compared with normal tissue targeting of its 30UTR. The inverse correlation between RhoA and around the tumor and nonmalignant breast epithelial cells (166). miR-146a expression in breast tumor cells was further confirmed Transient transfection of MDA-MB-231 and MCF-7 cells with through the transfection of the miR-146a vector in MDA-MB-231, miR-135b mimics showed that the proliferation, migration, and where the forced overexpression of this oncomiR significantly reduced colony formation of these cells were remarkably higher than those the RhoA expression in both gene and protein levels. As a result of transfected with the miR-135b inhibitor as well as the negative upregulation of miR-146a in MDA-MB-231, these cells lost their control (166). The cell-cycle assay also confirmed that miR-135b– motility and invasion ability, suggesting the suppressor effect of transfected cells showed a higher percentage of cell arrest in S and G2– miR-146a in breast cancer metastasis through the RhoA-dependent M phases in comparison with the control and miR-135b inhibitor pathway (152). groups. According to results from the Western blot analysis, increased Although the positive role of RhoA in the metastatic feature of protein expression of LATS2 in the MDA-MB-231 and MCF-7 cells breast tumor cells have been well confirmed, there is a study showing transfected with the miR-135b inhibitor leads to downregulation of that direct targeting of this protein by miR-155 promotes TGFb- CDK2 and p-YAP (166). induced EMT (153). In that study, Kong and colleagues found that the More recently, the role of a miRNA-HERC4 pathway has been expression level of miR-155 in invasive breast tumor cells was remark- confirmed in downregulation of LATS1 in breast cancer cells. Inves- ably higher than noninvasive one as well as normal breast cells. They tigation on the expression levels of miRNA-136-5p and miRNA-1285- reported that forced overexpression of miR-155 in the NMuMG cell 5p in different cancer cell lines, including MCF-7, BT474, and MDA- line leads to a significant decrease in cell invasion and migration. MB-231, showed that the expression of these two miRNAs are According to their results, TGFb/Smad directly upregulates miR-155 significantly lower in cancer cells as compared with the normal breast in the breast cancer cell (153). cell line (MCF-10A; ref. 167). miRNA-136-5p and miRNA-1285-5p Given that different members of GTPase family may participate in directly target the 30UTR of E3 ligase HERC4 that plays a critical role in the mechanotransduction signaling, future studies are required to ubiquitination and destabilization of LATS1 in cells. As a result of recognize other players and their related miRNA regulators in this downregulation of the aforementioned miRNAs in breast cancer cells, context. the expression of HERC4 increases, leading to a decrease in LATS1 expression (167). miRNA and the Hippo signaling TAZ is indirectly regulated by miRNA-125a through downregula- In recent years, the role of the Hippo signaling in cell behaviors has tion of leukemia inhibitory factor receptor (LIFR), which is an been investigated in various studies (154–156). The Hippo signaling upstream regulator of the Hippo signaling (168). Stem cells obtained pathway is responsible for controlling the nuclear accumulation of the from MCF-7, primary breast cancer cells, and MCF12A (nonmalig- YAP/TAZ effector that controls different biological events in cells such nant) cells, interestingly showed a different miRNA-125a expression as organ size, survival, and proliferation. The mechanical stimuli, in pattern. In this regard, the miRNA-125a expression level was found to addition to biochemical signals, influence the activation of the Hippo be significantly higher in stem cells obtained from breast cancer cells signaling pathway. The Hippo signaling pathway mediators mainly (MCF-7 and primary breast cancer cells) than those derived from the include YAP/TAZ, tumor suppressor 1/2 (LATS1/2), Mps One Binder nonmalignant MCF12A cell line (168). Further analysis showed a kinase activator (MOB1), mammalian Ste20-like kinases 1/2 (MST1/ lower LIFR protein level in stem cells derived from MCF-7 and 2), and SAV1 (157, 158). In brief, the activation of LATS1/2 by MST1/2 primary breast cancer cells when compared with MCF12A stem cells, occurs through phosphorylation at Thr1079/Thr1041 sites of LATS1/ indicating a reverse correlation between miRNA-125a and LIFR 2. Indeed, the SAV1–MST1/2 complex phosphorylates the MOB1 expression in breast cancer stem cells. Direct targeting of LIFR at the protein, which enhances its binding ability to LATS1/2, resulting in 30UTR site by miRNA-125a was confirmed through the luciferase phosphorylation and activation of LATS1/2. Finally, the activated reporter assay (168). As an upstream regulator of the Hippo signaling, LATS1/2 mediates YAP/TAZ phosphorylation, resulting in cyto- LIFR controls the stem cell pool dynamics. It was reported that forced plasmic accumulation and inactivation of the YAP/TAZ complex overexpression of miRNA-125a in MCF12A cells significantly through phosphorylation of YAP at Ser127 and binding to sequester increases the percentage of stem cells after 24 hours (168). In contrast, protein 14-3-3 (159). Studies on the function of the Hippo signaling in transfection of MCF-7 with miRNA-125a antagomir caused a signif- different cancer types have shown its important role in tumorigenesis. icant decrease in stem cell pool after the aforementioned time point. For example, in tumor microenvironment, YAP/TAZ activation Phosphorylation analysis of key regulators in the Hippo signaling enhances the ECM production by cancer-associated fibroblasts, lead- pathway exhibited an increase in the phosphorylation of LATS1 and ing to maintenance of cancer stem cells and their resistance to different TAZ in MCF-7 stem cells transfected by miRNA-125a. miRNA-125a cancer therapeutic approaches such as chemotherapy (160), radio- was suggested to activate the JAK2–STAT3 signaling by inhibiting of therapy (161), and various molecular targeted therapies (162). In phosphorylation of Hippo signaling mediators, promoting the non- general, overexpression of YAP/TAZ has been observed in various malignant breast epithelial stem cells toward malignant cells cancer types. A variety of studies indicated that the overactivation and (ref. 168; Fig. 5). expression of YAP/TAZ are associated with proliferation, invasion, In miRNA biogenesis, mature miRNAs are produced from pre- and metastasis of breast cancer cells (163). miRNA hairpins by Dicer enzyme (169). Chaulk and colleagues The role of miRNA in the regulation of the Hippo signaling is highly conducted a study to find the role of YAP/TAZ in total miRNA prominent, so that miRNA dysregulation was frequently found to be biogenesis in the MCF-10A cell line. According to their results, low correlated with downregulation or upregulation of Hippo signaling cell density of MCF-10A causes nuclear accumulation of YAP/TAZ

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SLIT2

JAM-A

ROBO1

miR-142-3p Syndecan-1

miR-362-3p/ Integrin-aV P130Cas miR-329

WASL GIT1 miR-10b miR-203 FAK miR-10b miR-490-3p/ miR-551a/miR-7

miR-146a miR-142-3p miR-149 Rho A Fascin

FHOD1 PPM1FA miR-18a TWF1 VIM BDNF PTEN miR-200c

miR-204 PHACTR1 miR-30c

HOXA9 miR-1285-5p

miR-136-5p/ miR-584 HERC4

miR-125a LATS1

LIFR

miR-135a

LATS2

Figure 5. The interaction between miRNAs and effectors involved in the mechanotransduction cell signaling in breast cancer. Various miRNAs control the expression of different effectors in the mechanotransduction signaling pathways including focal adhesion complex mediators, cytoskeleton binding proteins, and integrin proteins. Furthermore, miRNAs effectively control the expression of mediators in some mechanotransduction signaling pathways, including YAP/TAZ signaling. ECM stiffness also directly controls the expression of miRNAs in normal breast tissue cells. In response to ECM stiffness, the expression of some miRNAs, including miR-18a and miR- 203, changes that influences the cell behaviors. However, the exact mechanism by which the mechanical clues changes the expression of miRNAs remains unknown. WASL, WASP Like Actin Nucleation Promoting Factor; FAK, Focal adhesion kinase; GIT1, G protein-coupled receptor kinase-interacting protein; P130Cas, P130 Crk- associated substrate; RhoA, Ras homolog gene family member A; FHOD1, Formin Homology Domain Protein 1; PPM1F, Protein phosphatase, Mg2þ/Mn2þ–dependent 1F; BDNF, brain-derived neurotrophic factor; TWF1, Twinfilin actin binding protein 1; VIM, Vimentin; PHACTR1, phosphatase and actin regulator 1; LIFR, LIF Receptor Subunit Alpha; LATS1, Large Tumor Suppressor Kinase 1; LATS2, Large Tumor Suppressor Kinase 2; HERC4, HECT and RLD domain containing E3 ubiquitin ligase 4; JAM-A, junctional Adhesion Molecule-A; HOXA9: Homeobox protein Hox-A9; ROBO1, Roundabout Guidance Receptor 1. and increased biogenesis of miRNAs. In contrary, high cell density of reported to directly alter the miRNA expression in breast epithelial MCF-10A impairs miRNA biogenesis through Let-7–dependent cells (175). MCF-10A cells cultured on a stiff polyacrylamide-based reduction in Dicer levels. YAP/TAZ nuclear accumulation significantly membrane exhibited an increased expression of miR-18a compared suppresses the Let-7 mature miRNA through LIN28, as a main with those grown on a soft membrane. miR-18a decreases the expres- regulator of this protein (170, 171). The tumor suppressor role of sion of PTEN and HOXA9 genes, leading to induction of invasion and Let-7 has been well proved in various studies (172–174). metastasis through over-activation of the IP3/AKT signaling (175). Despite widespread efforts in recent years to further understand the It is critical for cells to regulate the expression of mechanotransduc- Hippo signaling pathway, there are still many questions about how this tion mediators to prevent their overactivation that disrupts cellular pathway interacts with other cell signaling pathways. miRNA dysre- homeostasis (176). One of the example in the context is regulation of gulation may indirectly affect the Hippo signaling through other the ROBO1 protein by miR-203. This protein is the key effector in the signaling pathways in breast cancer cells. However, more investiga- SLIT2/ROBO1 signaling in which the interaction between ROBO1 and tions will be helpful in this area. SLIT2 results in activation of adaptor proteins such as different members of the Rho GTPase family due to interaction with the ECM features and miRNA expression cytoplasmic side of ROBO1 proteins (177). Le and colleagues reported The relationship between ECM stiffness and breast cancer progres- that ECM stiffness directly affects the expression of miR-203 so that sion has been investigated in recent years. The ECM stiffness was the normal murine mammary gland cells (NMuMG) cultured on a

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high-density collagen gel exhibited lower expression of miR-203 and Therefore, understanding the key regulators that control the higher expression of ROBO1 compared with those grown on a low- mechanotransduction signaling will help provide a comprehensive density gel. They reported that miR-203 directly targets the 30UTR of road map in cancer therapy. miRNAs are probably to be the missing ROBO1 and downregulates its expression in response to physical piece of the puzzle that serves as the master regulators of mechan- features of ECM (178). otransduction signaling pathways. Recent advances in molecular Despite the fact that mechanical cues have an important role in gene technologies have made it possible to analyze the concurrent regulation mediated by miRNAs in cancer and normal breast cells, expression of many miRNAs and their possible targets in the cancer there are many unknown aspects due to the lack of comprehensive cells. Until now, the relationship between the dysregulation of studies in this area. In future, targeted investigations will help clarify miRNAs and changes in expression of proteins involved in the cell the mechanisms underlying how mechanical signals regulate the signaling pathway has been clarified in different studies. However, expression of miRNAs in breast cancer cells. some fundamental questions regarding how transcription factors affect the expression of miRNAs in response to the mechanical stresses remain unknown. Moreover, focusing on the role of Future Perspective mechanical forces in epigenetic modificationsofmiRNAgenesin In the past decades due to increased environmental risk factors, breast cancer cells that alter their expressions will increase our aging society and the complex lifestyle in modern society signifi- knowledge in this context. It is obvious that recognition of different cantly increased the rate of breast cancers. Therefore, research mediators involved in the regulation of miRNAs in response to councils and other funding bodies, including pharmaceuticals and physical forces will enable us to design effective therapeutic strat- other biomedical industries, invested billions of dollars in early egies to restore the expression of miRNAs to the normal state in diagnosis and effective treatment of breast cancer. To design a novel breast cancer cells. diagnostic tool and effective treatment, the scientific research focus has been on understanding the underlying mechanisms in breast Disclosure of Potential Conflicts of Interest cancer development. The role of physical stimuli is major factors in No potential conflicts of interest were disclosed. the development of tumors, in this context, so that cell stress signals within the tumor microenvironment affect the different phases of Received December 20, 2019; revised March 14, 2020; accepted May 15, 2020; cancer development such as proliferation, invasion, and metastasis. published first May 19, 2020.

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Key Regulatory miRNAs and their Interplay with Mechanosensing and Mechanotransduction Signaling Pathways in Breast Cancer Progression

Hamid Najminejad, Behrouz Farhadihosseinabadi, Mehran Dabaghian, et al.

Mol Cancer Res 2020;18:1113-1128. Published OnlineFirst May 19, 2020.

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