Regulating Signaling in Breast Cancer
MicroRNAs (miRNAs) are small non-coding RNAs that fine-tune the expression of target mRNA transcripts at the post-transcriptional level mainly by direct interaction with their 3’UTR. They frequently regulate multiple related target genes acting on the same phenotype or even within the same signaling pathway. Thereby, they are commonly involved in cancer-related phenotypes such as proliferation, apoptosis or cell motility. Here we will review the last insights about microRNAs regulating signaling in breast cancer with the focus on EGFR and NF-κB signaling, two pathways which are frequently de-regulated in mammary neoplasms and contribute to disease progression.
Cell signaling is a complex molecular process that controls diverse biological functions of the cells and their intercellular communications and is pivotal for fundamental cellular functions including proliferation, differentiation and apoptosis. The signals are initiated mostly in an extrinsic manner by binding of growth factors, cytokines or other ligands to the cognate receptors expressed on the target cell. Stimulation of such receptors leads to a series of downstream biochemical reactions, where frequently different cascades of protein kinases are activated. Eventually, these continuous events lead to the activation of specific transcription factors in order to control and maintain different genetic programs. Intrinsic regulatory mechanisms (e.g. feedback and feedforward loops) are applied in order to maintain the balanced function of signaling pathways by determining the duration, the amplitude or the frequency of a signal. However, alterations leading to hyper- or hypo-activation of these signaling pathways have been connected with various human diseases, including cancer.
Breast cancer is the most frequent type of cancer in women (23% of all cancers) and is still the second leading cause of cancer mortality in women worldwide (14% of all cancer deaths), as delineated by the World Health Organization database (Globocan).
As a disease, breast cancer is heterogeneous with respect to clinical outcome, cellular composition and molecular alterations. Based on molecular gene expression signatures, breast cancer can be classified into five different subtypes (luminal A, luminal B, basal-like, ERBB2- positive and normal-like) which differ in their repertoire of de-regulated signaling pathways . For instance, NF-κB signaling, as well as EGFR signaling are frequently constitutively activated in the aggressive basal-like or triple-negative and ERBB2/HER2-positive subtypes of breast cancer [2,3]. Such alterations in signal transduction may contribute to breast cancer initiation, progression and metastasis [4,5].
MicroRNAs are endogenous small non-protein- coding RNAs of ~22 nucleotides in length and are highly conserved in the genomes of animals, plants, fungi and viruses. They constitute a large class of negative regulators of gene expression mainly by base pairing with the 3’-untranslated region (3’-UTR) of their target messenger RNA (mRNA), with perfect or nearly perfect complementarity . However, miRNA interaction has also been reported with the open reading frame (ORF) and the 5’-UTR of the target genes [7,8]. The mature miRNAs can negatively regulate gene expression through different mechanisms including mRNA degradation, translational inhibition or mRNA deadenylation . It is estimated that miRNAs account for approximately 1% of the expressed human genome and they can target more than 30% of the protein-coding genes [10,11]. Hence, it is not surprising that miRNAs have been found to play a role in different biological processes including embryogenesis and stem cell maintenance, apoptosis, and aging [12-14]. Notably, their expression has been found to be deregulated in a wide range of human diseases including neurodegenerative diseases and cancer [15,16].
MicroRNAs as Oncogenes or Tumor Suppressor Genes
As cancer is ultimately a consequence of disordered gene expression and miRNAs are often deregulated in tumors, miRNAs have been suggested to contribute to the development of cancer by modulating the levels of critical proteins during this process. MicroRNAs can act either as oncogenes or tumor suppressors, depending on the cellular context and on the target genes they regulate. In breast cancer, the best characterized oncogenic miRNAs are miR-21, miR-181a, miR- 181b, and miR-155. MiR-21 is frequently upregulated in the majority of breast tumors and its expression is inversely correlated to PDCD4 and PTEN expression [17-19]. In breast cancer cells, PTEN repression by miR-21 induces the activation of AKT1, an upstream activator of NF-κB signaling and thus, contributes to enhanced tumorigenesis . The pro-apoptotic protein Bim and the tumor suppressor Cylindromatosis gene CYLD were identified as miR-181a and miR-181b targets, respectively [18,20]. MiR-181b was identified as a key player in a positive feedback loop linking inflammation to an epigenetic switch that controls cellular transformation in human mammary epithelial MCF-10A cells and a negative regulator of CYLD, a deubiquitinating enzyme that negatively regulates NF-κB signaling . On the other hand, the TGF-β induced miR- 181a was recently characterized as “metastamiR” as it was found to enhance the metastatic potential of breast cancers by promoting epithelial- mesenchymal transition, migratory, and invasive phenotypes. Along these lines, miR-181a expression was dramatically upregulated in metastatic breast tumors, and particularly in triple- negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients . The fact that miR- 155 has an oncogenic function is supported by Jiang and colleagues showing that this miR is overexpressed in tumors, promotes tumor growth in vivo and its expression is inversely correlated with the expression of the tumor suppressor SOCS1 in breast cancer cells .
Among the miRNAs with tumor suppressive function are let-7, miR-7, miR-200c, miR-31, miR-146, miR-124, miR-125a/b and miRG 373/520 family. Several studies indicate that let- 7 can act as an anti-tumorigenic factor . Namely, lenti-viral introduction of let-7 into breast cancer initiating tumor cells reduced their proliferation, mammosphere formation and metastasis formation in vivo . Let-7 was also shown to be implicated in NF-κB signaling activation by suppressing the pro-inflammatory cytokine interleukin-6 (IL-6) and RAS oncogene during transformation of mammary epithelial cells . However, the current knowledge on the miRNA regulation of NF-κB signaling seems to be more complex than initially thought. MiR- 146 was initially identified as a transcriptional target gene of NF-κB in lipopolysaccharide (LPS)-induced human monocytes . Nevertheless, it was later shown that this miR can also function in a negative feedback loop by suppressing the expression of the NF-κB upstream activators IRAK1 kinase and TRAF6, also in the context of breast cancer cells . Further, Koerner and colleagues described that miR-31, a miR that its expression is almost abolished in advanced breast tumors, inhibits NF-κB activity by targeting the protein kinase C epsilon (PRKCE), and thus resulting in enhanced sensitivity of breast cancer cell lines towards ionizing radiation as well as chemotherapeutic drugs . MiR-7, miR-200c and miR-125a/b have been described as important inhibitors of the epidermal growth factor receptor signaling (EGFR), an oncogenic pathway highly relevant in breast tumors, as it supports tumor growth and progression. In particular, miR-7 and miR-125a/b have been reported to directly target the EGFR and the ERBB2 receptor genes [27,28], respectively, while miR-200c controls cell cycle progression and the invasive capacity of breast cancer cells by regulating PLCγ .
Interestingly, individual miRNAs can target multiple genes and each protein-coding gene can be regulated by several miRNAs, thus adding one more level of complexity to these gene networks. Uhlmann and colleagues reported that multiple miRNAs may function in a combinatorial manner in order to control EGFR signaling pathway at different levels. As oncogenic pathways are often resistant to the inhibition of individual regulators, by using this network-analysis approach, a set of a few miRNAs whose combined activity may be strong enough to inhibit important oncogenic pathways could be applied to treat breast tumors. Moreover, this approach led to the identification of miR-124, miR-147 and miR-193a-3p as novel tumor suppressors that co-target EGFR-driven cell-cycle network proteins and inhibit cell-cycle progression and proliferation in breast cancer . Last but not least, individual miRNAs may also target multiple genes within different pathways, and thus orchestrate the fine-tuning of different signal networks towards a certain phenotype. For instance, miR-373/520 family was recently shown to inhibit both NF-κB and TGF-β signaling pathways, by simultaneously targeting RELA and TGF-β receptor 2 (TGFBR2) leading to reduced cell invasion in vitro and in vivo, specifically in estrogen receptor (ER) negative breast tumors . To conclude, these findings suggest that specific miRNAs may have a role beyond the tumor- initiating event and directly participate in tumor progression and metastasis, by regulating critical cancer-related genes and signaling pathways. An illustration of some examples of the complex miRNA regulation of signaling pathways in breast cancer is given in figure 1.
During the last decade, a fundamental role of miRNAs to virtually all cancer-relevant signaling pathways has been established. As reviewed above, introducing synthetic tumor-suppressive miRNAs (e.g. miR-31, miR-520c, miR-200c) into breast cancer cells has proven a powerful tool to inhibit oncogenic phenotypes such as cell invasiveness, metastasis, enhanced proliferation or resistance to apoptosis [26,29,30]. Hence, ectopic expression of these miRNAs specifically within tumors might provide a novel strategy towards treatment of breast cancer by interfering with multiple synergistic signaling pathways at the same time. Further, the intense study of the target spectra of such highly efficient tumorsuppressive miRNAs might facilitate the design of novel therapeutic approaches mimicking the effect of miRNA overexpression on the activity of cancer-related signaling pathways.
 Sorlie T.: PNAS 98, 10869–10874 (2001)
 Biswas D. K. et al.: PNAS 101, 10137–10142 (2004)
 Kenny P. A. and Bissell M. J.: Journal of Clinical Investigation 117, 337–345 (2007)
 Sovak M. A. et al.: Journal of Clinical Investigation 100, 2952–2960 (1997)
 Spencer K. S. R.: The Journal of Cell Biology 148, 385–397 (2000)
 Gu S. et al.: Nature Structural & Molecular Biology 16, 144–150 (2009)
 Lewis B. P. et al.: Cell 120, 15–20 (2005)
 Lee I. et al.: Genome Res. 19, 1175–1183 (2009)
 Esquela-Kerscher A. and Slack F. J.: Nature Reviews Cancer 6, 259–269 (2006)
 Lim L. P. et al.: Science 299, 1540 (2003) Lewis B. P. et al.: Cell 115, 787-798 (2003)
 Bernstein E. et al.: Nat. Genet. 35, 215-217 (2003)
 Cheng A. M. et al.: Nucleic Acids Res. 33, 1290-1297 (2005)
 Liang R. et al.: Curr. Genomics 10, 184-193 (2009)
 Maes O. C. et al.: Curr. Genomics 10, 154-168 (2009)
 Calin G. A. et al.: Proc. Natl. Acad. Sci. U.S.A. 99, 15524-15529 (2002)
 Iorio M. V.: Cancer Research 65, 7065-7070 (2005)
 Iliopoulos D. et al.: Mol. Cell 39, 493-506 (2010)
 Zhu S. et al.: Cell Research 18, 350-359 (2008)
 Taylor M. A. et al.: Journal of Clinical Investigation 123, 150-163 (2012)
 Jiang S. et al.: Cancer Research 70, 3119-3127 (2010)
 Yu F. et al.: Cell 131, 1109-1123 (2007)
 Iliopoulos D. et al.: Cell 139, 693-706 (2009)
 Taganov K. D. et al.: Proc. Natl. Acad. Sci. U.S.A. 103, 12481-12486 (2006)
 Hurst D. R. et al.: Cancer Research 69, 1279-1283 (2009)
 Koerner C. et al.: J. Biol. Chem. (2013). doi:10.1074/jbc.M112.414128
 Scott G. K. et al.: J. Biol. Chem. 282, 1479-1486 (2007)
 Uhlmann S. et al.: Molecular Systems Biology 8, (2012)
 Uhlmann S. et al.: Oncogene 29, 4297-4306 (2010)
Keklikoglou I. et al.: Oncogene 31, 4150-4163 (2012)