Journal of Biochemistry Advance Access published April 9, 2015
JB Review
MicroRNAs as the fine-tuners of Src oncogenic signaling
*Chitose Oneyama and Masato Okada
Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
*Correspondence should be addressed to Chitose Oneyama, PhD. Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Tel: 81-6-6879-8299
Fax: 81-6-6879-8298
e-mail:
[email protected] © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1
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Running title: microRNA controls c-Src-mediated tumor progression
Summary The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumor progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumor suppressors by regulating the malignant expression of signaling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signaling in various cancers. We summarize recent findings on Src-related
cancer therapeutics.
Key words cancer signaling, microRNA, mTOR, proto-oncogene, Src
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miRNAs, their target genes, mechanisms behind their interplay, and their implications for
Src is the first discovered oncogene and its product is first identified as non-receptor cytoplasmic tyrosine kinase (1). In many human neoplasms, including colorectal, breast, prostate, pancreatic, head and neck, and lung carcinoma; glioma; and melanoma, Src is overexpressed and/or hyperactivated (2). In fact, its dysregulation is a major oncogenic signature found in cancer (3). A large body of evidence has clearly demonstrated that Src is a key molecule in tumor progression (3, 4); therefore, Src has emerged as a promising molecular target for anti-cancer therapies (5, 6). Small-molecule inhibitors of Src that target the ATP- (e.g., dasatinib, nosutinib, and saracatinib) and substrate-binding sites (e.g., KX01) are currently
Conversely, Src participates in the maintenance of normal cell homeostasis and has a large range of physiological functions, including cell proliferation, survival, cytoskeletal organization, intercellular contact, matrix adhesion dynamics, migration, and motility (8). Once activated, Src acts as a common relay point for several downstream cascades originating from extracellular signals, such as growth factors and integrins, to intracellular signaling pathways (9). Thus, Src activity is strictly controlled via several mechanisms. The kinase activity of Src is negatively regulated by the phosphorylation of a regulatory Tyr in its C-terminal tail, catalyzed by C-terminal Src kinase (Csk) (10). Cysteine residues of Src are important for its stability and Src-induced cellular transformation (11). The E3 ubiquitin-ligase Cbl and Cullin-5 mediate the ubiquitination of Src and induce its degradation via the ubiquitin−proteasome pathway (12, 13). In normal cells, Csk suppresses Src transformation potential even when Src is abundantly overexpressed (10). Thus, many studies have exploited viral Src (v-Src), which can induce a full transformed
phenotype,
including
increased
proliferation,
anchorage-
and
growth
factor-independent growth, and refractile cell morphology (14). However, there are doubts whether v-Src demonstrates the intrinsic function of cellular Src (c-Src) because highly active v-Src induces aberrant phosphorylation of various proteins (3, 4). Although the Src gene is rarely mutated and is expressed as wild-type c-Src in human cancers (15), a recent study reported that the copy number of c-src was increased in colorectal cancer (16). This indicates
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undergoing clinical trials (7).
that the breakdown of Src regulatory mechanisms mainly accounts for its increased expression, activity, and subsequent malignant phenotypes, such as uncontrolled mitogenicity, enhanced survival, and invasive ability, in cooperation with other oncogenic signals. To verify the function of c-Src in cancer progression, an experimental system using Csk-deficient mouse embryonic fibroblasts (Csk−/−MEF) in which c-Src could induce cellular transformation beyond the control of Csk was developed (17). In this system, the activation of a relatively small numbers of molecules is sufficient for the induction of cellular transformation and tumorigenesis. In other words, this system could be useful in the identification of critical
phosphorylated Cbp/PAG binds to activated c-Src, causing c-Src to translocate into lipid rafts, and that sequestration of c-Src in lipid rafts is sufficient to suppress c-Src-mediated transformation (Figure 1) (18, 19). Recently, this in vitro system was utilized to examine the contribution of microRNAs (miRNAs) to c-Src-mediated tumor progression. miRNAs are a family of small, endogenous, and evolutionarily conserved non-coding RNAs involved in the regulation of essential, cellular, and functional processes, including proliferation, differentiation, survival, and stress responses. miRNA genes are transcribed as pri-miRNA and processed to pre-mRNA by Drosha/DGCR8. The pre-miRNA is then cleaved by Dicer to yield an approximately 22-bp duplex wherein one strand is incorporated into the RNA-induced silencing complex as a mature miRNA. When a miRNA binds its target mRNA, it either regulates protein translation or induces degradation of the target mRNA (20). To date, more than 2000 mature miRNAs have been identified in humans, and the number of miRNA has been growing. miRNAs diversely regulate gene expression; a single miRNA can target multiple target genes or a single gene can be targeted by multiple miRNAs (21). This interplay between miRNA and transcripts underscores the potential role of miRNAs as key regulators of complex signaling networks (22). Recently, it was shown that numerous miRNAs are extensively dysregulated in several human diseases, including cancer (23). miRNAs are known to play key roles in many types of cancers, upregulated and/or
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pathways leading to c-Src transformation. Using this model system, we found that
downregulated, and to function as oncogenes or tumor suppressors (24). Indeed, many miRNAs are found in cancer-related genomic regions or fragile sites. Thus, their expression profiles have been vigorously investigated and can be used for classification, diagnosis, and prognosis of different cancer types (25). Furthermore, miRNA profiling may provide novel signaling axes and new approaches for the manipulation of cancer-related genetic pathways because miRNA can modulate many genes and fine-tune cell signaling. In this review, we highlight recent studies and our findings on Src-related miRNAs involved in several human cancers (Table 1).
Using the system described above, the expression profiles of miRNAs between non-transformed and c-Src-transformed cells were compared (26). Microarray profiling revealed that c-Src activation regulates a limited set of miRNAs; seven miRNAs were downregulated and six were upregulated more than 2-fold. It is indicated that miRNAs act as tumor suppressor when their expressions are downregulated. In fact, many miRNAs are silenced in human cancers by methylation, loss of heterogeneity, or for other reasons (27). In the Src-transformed cells, downregulated miRNAs are categorized into three clusters based on their chromosomal locations: miR-23b and miR-27b (mouse chromosome 13), miR-322 (-424 in human), miR-450a, miR-503, and miR-542 (X chromosome), and miR-99a (chromosome 16). A colony formation assay in soft agar revealed that among seven miRNAs, five (miR-27b, miR-99a, miR-322, miR-503, and miR-542) dramatically suppressed anchorage-independent growth and tumorigenesis in c-Src-transformed cells injected into nude mice. The studies on the function of each of these five miRNAs uncovered miRNA-mediated c-Src oncogenic signaling and crosstalk between Src and other oncogenic signaling pathways, such as mammalian target of rapamycin (mTOR) and focal adhesion-mediated pathways (Figure 2).
miRNA-mediated link between Src and mTOR in cancer progression
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miRNA-mediated Src oncogenic signaling
The evolutionarily conserved Ser/Thr kinase mTOR is a major downstream effector of the phosphoinositide-3-kinase (PI3K) pathway and plays pivotal roles in regulating cell growth, proliferation, and survival (28). mTOR assembles with alternative binding partners to generate two functionally distinct protein complexes: mTOR complex 1 (mTORC1) containing Raptor and mTOR complex 2 (mTORC2) containing Rictor (29). mTORC1 controls cell growth by regulating mRNA translation via phosphorylation of downstream substrates, ribosomal S6 kinase, and 4E binding protein 1 (30). By contrast, mTORC2 regulates cell proliferation, survival, and the actin cytoskeleton by activating protein kinase B (also called AKT), protein
signaling is frequently observed in many types of cancers, implicating it in the promotion of tumor growth and malignancy (32). Indeed, rapalogs, selective mTORC1 inhibitors, have been clinically administered in advanced kidney cancer, and mTOR kinase inhibitors, such as Torin, have been developed as anti-cancer drugs for several types of cancer, such as those of the pancreas, stomach, colon, and breast (33). Recently, the mechanisms underlying Src-mediated activation of mTOR signaling via miRNA expression in cancers were demonstrated.
miR-99a−mTOR MiR-99a is downregulated in various human cancers, such as ovarian carcinoma, squamous cell carcinoma of the tongue, and bladder cancer. Furthermore, the miR-99a gene is located in the commonly deleted region at 21q21.1 in human lung cancer (34). We found that miR-99a is downregulated in Src-transformed cells and that re-expression of miR-99a suppressed tumor growth of these cells (26). We showed that miR-99a targets mTOR and fibroblast growth factor receptor 3 (FGFR3), both of which are tightly associated with human cancers. FGFR3 promotes cell survival by stimulating PI3K−AKT signaling (35). Functional analysis showed that miR-99a expression is regulated by Src-related oncogenic pathways involving epidermal growth factor receptor (EGFR), c-Src, K/H-Ras, and mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). MiR-99a downregulation is associated with
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kinase C-α and serum glucocorticoid-induced protein kinase-1 (31). Dysregulation of mTOR
mTOR/FGFR3 upregulation in various human lung cancer cells/tissues; in fact, the introduction of miR-99a to these cells suppressed tumorigenicity. This study demonstrated that the Src pathway−miR-99a−mTOR/FGFR3 axis is crucial for controlling tumor growth in a wide range of human cancers and indicated that miR-99a is a missing link between Src and mTOR, which have both been correlated with human cancer. MicroRNA-mediated mTOR regulation has also been shown in studies of miR-100 and miR-199-3p (36, 37). MiR-100 contains the same seed sequences as miR-99a and is downregulated in human cytomegalovirus infection, clear cell ovarian cancer, and childhood
Taken together, these studies highlight a critical role of miRNA-regulated mTOR signaling in controlling tumor growth in various human cancers.
miR-424/503−Rictor In human colon and prostate cancers and Src-transformed cells, we found that expression of Rictor, an exclusive component of mTORC2, is controlled by the miR-424/503 cluster, which is downregulated in Src-transformed cells (39). Re-expression of miR-424/503 suppressed tumorigenicity and invasiveness of cancer cells by affecting cytoskeletal organization and formation of focal adhesions. Rictor upregulation via repression of the miR-424/503 cluster induced formation of mTORC2 and subsequent activation of AKT and promoted tumor growth and invasive activity of cancer cells. Downregulation of miR-424/503 is associated with Rictor upregulation in human colon cancer tissues. This study shows that the miR-424/503−Rictor pathway plays a crucial role in tumor progression. Moreover, Rictor is also regulated by miR-218 and miR-152 in oral squamous cell carcinoma and endometrial cancer (40, 41). Taken together, these data indicate that miRNA-mediated Rictor upregulation contributes to tumor progression in a wide array of human cancers. MiR-424/503 comprise a polycistronic gene cluster, and both miRNAs target Rictor by recognizing different regions of the Rictor 3′-untranslated region (39). Thus, the formation of
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adrenocortical tumors (36, 38). MiR-199-3p is downregulated in hepatocarcinoma cells (37).
miRNA clusters whose members target common genes would exert an amplified effect on the mRNA targets of individual miRNAs. Notably, miR-424/503-mediated Rictor upregulation can collaborate with miR-99a-mediated mTOR upregulation to promote mTORC2 activation under the control of Src-related oncogenic signaling.
miRNA-mediated control of focal adhesion and Src activation Earlier studies have suggested that highly metastatic cancer cells display elevated c-Src kinase activity, which is further enhanced by extracellular stimuli (2, 3). In cell transformation
ERK, and AKT, can be activated by integrin-mediated cell−extracellular matrix (ECM) adhesion (42, 43). Although aberrant signaling has been studied at the point where phosphorylation/dephosphorylation regulatory mechanisms are disrupted, we suggest that miRNA mediates the regulation of focal adhesion and activation of downstream effectors in Src-activated cancer cells.
miR-542-3p−ILK Integrin-linked kinase (ILK) is a Ser/Thr kinase and adaptor protein localized to focal adhesions (44). ILK mediates intracellular signaling for actin cytoskeletal reorganization, cell migration, proliferation, survival, invasion, and epithelial-to-mesenchymal transition (EMT) via integrins and growth factor receptors. Once ILK is activated by cell−ECM and growth factor receptors, ILK phosphorylates and activates glycogen synthase kinase 3β and AKT (45). Although ILK overexpression and deregulation are frequently observed in several human cancers (45, 46), the mechanism of ILK upregulation in cancer cells is poorly understood. We found that ILK is targeted by miR-542-3p, a downregulated miRNA in Src-transformed cells (47). MiR-542-3p is downregulated by Src-related oncogenic pathways, such as EGFR, Ras, and MEK/ERK. Ectopic expression of miR-542-3p suppresses tumor growth and more effectively, invasiveness of human colon cancer cells. ILK upregulation by the repression of
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and tumor progression, cell signaling, including focal adhesion kinase (FAK), Src family kinase,
miR-542-3p promotes cell adhesion and invasion by activating the integrin−FAK/Src pathway and also induces AKT/glycogen synthase kinase 3β-mediated cell survival. Downregulation of miR-542-3p is significantly correlated with upregulation of c-Src and ILK; there is also a correlation between ILK upregulation and c-Src activation in human colon cancer tissues. Furthermore, it was found that miR-542-3p-mediated ILK downregulation induces inactivation of c-Src and FAK in human colon cancer cells. These lines of evidence suggest that upregulation of c-Src activity is induced via a miR-542-3p−ILK-mediated positive feedback loop that can be initiated by oncogenic signaling by growth factors and integrins.
MiR-27b is downregulated in various human cancers, including colon, lung, breast, and prostate cancers (48, 49). We found that miR-27b is under the control of the PI3K pathway, implying that miR-27b is downregulated in a wide range of human cancers harboring PI3K upregulation. Re-expression of miR-27b in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasive ability. MiR-27b controlled tumor growth by targeting ARFGEF1 and more importantly, suppressed cell−ECM adhesion and invasiveness of cancer cells by targeting paxillin. Paxillin is a critical component of the focal adhesion complex that is phosphorylated by c-Src and FAK upon adhesion via integrins (50). Paxillin serves as a platform for adaptor proteins that activate downstream intracellular signaling (51). Paxillin is upregulated in various human cancers and is involved in tumor malignancies (52, 53). We also found that miR-27b-mediated downregulation of paxillin attenuates focal adhesion-mediated Src activation and in turn, suppresses miR-27b repression. This study showed that miR-27b-mediated paxillin upregulation promotes c-Src signaling evoked by integrin-mediated cell adhesion, resulting in the promotion of malignant phenotypes, such as invasion and metastasis (submitted).
Other miRNAs that regulate downstream effectors of Src signaling
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miR-27b−Paxillin
miR-29b−ID1 Src regulates the invasion of cancer cells through inhibitor of DNA binding/differentiation 1 (ID1). ID1 is frequently overexpressed in human cancer and is associated with tumor malignancies (54). ID1 expression is accompanied by the overexpression of Src and matrix metallopeptidase 9, a downstream effecter of ID1, in human lung adenocarcinoma. MiR-29b was identified as a miRNA involved in Src−ID1 signaling by treatment with saracatinib, a Src inhibitor, for lung cancer (55). MiR-29b targeted ID1 and reduced the migration and invasion of lung cancer cells. Repression of miR-29b-mediated ID1 downregulation diminished the effect
represses miR-29b through β-catenin and c-Myc signaling, suggesting that Src cooperates with the c-Myc pathway to induce ID1 expression by suppressing miR-29b. They also suggest that the Src−miR-29b−ID1 pathway is important for controlling lung cancer malignancies and is a potential predictive marker for Src kinase inhibitors.
miR-30−TMPRSS2-ERG Src is important for the development of bone metastasis and the castration resistance of prostate cancer (56). Because Src inhibitors are being tested in clinical trials for the treatment of patients with prostate cancer (57), Kao et al. identified miRNAs that respond to Src inhibitors in prostate cancer (58). miRNA expression profiles show that miR-30 family members are most prominently upregulated by Src inhibitor treatment. MiR-30 targets the erythroblast
transformation-specific-related gene (ERG), and its overexpression in prostate cancer cells suppressed EMT, cell migration, and invasion. ERG is a frequently overexpressed oncogene that is translocated as TMPRSS2-ERG fusion transcripts. Although TMPRSS2-ERG is regulated by androgens at the transcriptional level, Kao et al. showed that ERG upregulation is modulated via miR-30 by Src activation. Their study also revealed a novel crosstalk between Src and androgen signaling through miRNA.
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of this Src inhibitor on migration and invasion. Rothschild et al. showed that activated Src
miR-126−Crk Li et al. examined the role of miRNA in Src transformed mouse embryonic brain cells (59). Out of the nine miRNAs affected by v-Src, upregulation of miR-224 and downregulation of miR-126 were responsible for promoting anchorage-independent growth and migration, respectively. Expression of miR-126 suppressed motility and invasive activity of human mammary carcinoma cells by targeting Crk, a component of focal adhesion (60). Because Src phosphorylates Crk-associated substrate (Cas), this study demonstrates that Src coordinately regulates the Crk/Cas complex to promote tumor cell growth and invasion by two independent
control of miRNA expression thereby increasing Crk expression.
miRNA-mediated regulation of Src expression and tumor progression In previous sections, miRNAs regulated downstream of Src signaling were summarized. These miRNAs are involved in cancer progression and invasion via perturbed expression of their target genes. Recent studies showed that the expression of Src is also disrupted via several miRNAs in cancer cells. Dysregulation of these miRNAs and those downstream of Src signaling jointly promote tumor malignancy. Moreover, it is reported that they partly account for resistance against drugs targeted to Src signaling.
miR-23b MiR-23b is a mediator of multiple metastatic steps, including tumor growth, invasion, and angiogenesis, although it functions as a tumor suppressor or oncomir in different cell types (61). MiR-23b is downregulated in various human cancers, including bladder, prostate, pancreatic, and breast cancers. In prostate cancer, miR-23b is silenced by methylation and targets c-Src. miR-23b and miR-27b are encoded by the same gene cluster, miR-23b~27b~24-1, suggesting that the expression of both miR-23b and miR-27b is regulated through a common pathway. Some studies show that miR-23b and miR-27b are downregulated in human castration-resistant
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mechanisms: 1) Src phosphorylation of Cas that activates Cas-related signaling and 2) Src
prostate cancers (62). We also observed that the expression of miR-23b and miR-27b was repressed by c-Src-induced transformation. Taken together with the miR-27b−paxillin axis described above, it is possible that c-Src is regulated by the miR-23b~27b~24-1 gene cluster via dual mechanisms: regulation of c-Src kinase activity via miR-27b-mediated upregulation of paxillin and regulation of paxillin mediated by miR-23b. Furthermore, because the expression of both miR-23b and miR27b is downregulated by c-Src, upregulation of c-Src expression and activity may amplify the positive-feedback loop mediated by the miR-23b~27b~24-2 gene cluster, thereby promoting tumor malignancy mediated by c-Src activity.
In renal cell carcinoma (RCC), Src contributes to the appearance of malignant phenotypes, such as angiogenesis, through Src−STAT3−vascular endothelial growth factor signaling (63). Malid et al. showed that miR-205 was significantly suppressed in RCC lines/tissues, and overexpression of miR-205 suppressed cell proliferation, migration, and invasion in renal cancers cell (64). The effect of miR-205 is achieved by the reduction of Src-family kinases, including Src, Lyn, or Yes, and is mediated by the Src−ERK pathway. Because 30% of RCC patients develop metastatic recurrence, identification of biomarkers for RCC to better predict cancer development and prognosis is necessary. MiR-205 would be a potential therapeutic target in the treatment of RCC and a good biomarker for RCC diagnosis. Resistance of non-small cell lung cancers (NSCLC) against EGFR tyrosine kinase inhibitors (TKI), such as erlotinib, involves the induction of EMT and Src activation (65, 66). CRIPTO1, an epidermal growth factor-CFC family member, is overexpressed in a variety of human cancers (67). Park et al. revealed that CRIPTO1 renders EGFR-TKI-sensitive and EGFR-mutated NSCLC cells resistant to erlotinib (68). CRIPTO1 activates Src to stimulate downstream pathways through the downregulation of miR-205 in EGFR-mutant lung cancer cells resistant to erlotinib. Indeed, expression of CRIPTO1 correlated with EGFR-TKI resistance in lung cancer patients harboring EGFR-sensitizing mutations. This study suggested the efficacy of
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miR-205
co-targeting EGFR and Src signaling for the treatment of EGFR-TKI-resistant, CRIPTO1+, and EGFR-mutated NSCLC patients.
miR-31 Downregulation of miR-31, which is observed in melanoma tumors, is associated with genomic loss of the region in chromosome 9q21 that encompasses miR-31 or with epigenetic silencing by DNA methylation and EZH2-mediated histone methylation (69-71). Asangani et al. reported that the overexpression of miR-31 suppressed migration and invasion of melanoma
targets enhance EZH2 activity, thereby resulting in further suppression of miR-31 expression and subsequent cancer progression.
miR-203 Src protein levels are consistently upregulated in lung cancer tissues (73). Wang et al. showed that miR-203 directly suppressed Src expression and consequently triggered the suppression of downstream signaling pathways, such as the Ras/ERK (74). They showed an inverse correlation between miR-203 and Src protein levels in lung cancer tissues. Because the restoration of Src expression can successfully attenuate the tumor suppressive effects of miR-203 on lung cancer cells, miR-203 mainly targets Src to exert its tumor-suppressive function during lung cancer progression. MET overexpression is involved in the resistance of NSCLCs to TKIs, such as gefitinib (75, 76). Garofalo et al. showed that miR-103 and miR-203 are upregulated by MET silencing and contribute to gefitinib sensitivity by inducing apoptosis and reducing EMT of gefitinib-resistant NSCLCs (77). Enforced expression of miR-103 or miR-203 increased the sensitivity of NDCLCs to gefitinib.
Perspectives
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cells (72). They identified Src, Met, Nik, and Rab27a as direct targets of miR-31, and these
Although many findings indicate that miRNAs can be useful biomarkers to diagnose cancers, mechanisms behind aberrant miRNA profiles in cancer and their role in cancer phenotypes need to be established. Recent studies have shed light on multiple miRNAs that work behind the scenes to regulate Src signaling. Signaling molecules, such as Src and mTOR, are linked to each other, not only via phosphorylation and inter-protein interactions but also via translational regulation by miRNAs. This interplay may lead to novel insights into cancer progression, invasiveness, and drug resistance. Furthermore, targeted molecular therapy against Src-related signaling molecules and Src-related miRNAs as biomarkers could provide novel
Acknowledgements We appreciate that this work was supported by The Exciting Leading-Edge Research Project at Osaka University (Japan) to CO; Grants-in-Aid for Scientific Research (B) to CO; and Grant-in-aid for P-DIRECT from the Ministry of Education Culture, Sports, Science and Technology of Japan to CO.
Conflicts of interest None declared.
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integrated therapeutic and/or diagnostic approaches to cancer.
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Table 1. miRNAs involved in Src-mediated cancer signaling Targets
Tumor
Ref
miR-99a
mTOR, FGFR3
lung
26
miR-424/503
Rictor
colon, prostate
39
miR-542-3p
ILK
colon
47
miR-27b
Paxillin
colon
submitted
miR-29b
ID1
lung
55
miR-30
TMPRSS2-ERG
prostate
58
miR-126
Crk
breast
59
miR-23b
Src
prostate
62
miR-205
Src
kidney
64
miR-31
Src
melanoma
72
miR-203
Src
lung
74
22
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miRNA
Figure Legend Figure 1. Regulation of c-Src function in cancer progression. Figure 2. Oncogenic signaling pathways which are regulated via miRNAs downstream of c-Src.
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23
Lipid rafts
Active
SH2
se
kina
P
Inactive
"$%
SH3
P P P
"&' P
Tumor Progression
Proliferation Survival Invasion/Migration/Adhesion Angiogenesis
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RTK integrins
P
FGFR3
integrin
EGFR
Focal Adhesion
paxillinFAK
c-Src
ILK
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!"#
GSK3! PI3K
miR-27b miR-542-3p
AKT
miR-99a miR-424/503
Tumor Progression mTOR Rictor
mTORC2