MicroRNA-based Therapeutic Strategies for Targeting Mutant and Wild Type RAS in Cancer

Abstract

MicroRNAs (miRs) have been causally implicated in the progression and development of a wide variety of cancers. miRs modulate the activity of key cell signaling networks by regulating the translation of pathway component proteins. Thus, the pharmacological targeting of miRs that regulate cancer cell signaling networks, either by promoting (using miR-supplementation) or by suppressing (using anti-sense oligonucleotide based strategies) miR activity is an area of intense research. The RAS-Extracellular signal regulated kinase (ERK) pathway represents a major miR-regulated signaling network that endows cells with some of the classical hallmarks of cancer, and is often inappropriately activated in malignancies by somatic genetic alteration through point mutation or alteration of gene copy number. In addition, recent progress indicates that many tumors may be deficient in GTPase activating proteins (GAPs) due to the collaborative action of oncogenic microRNAs. Recent studies also suggest that in tumors harboring a mutant RAS allele there is a critical role for wild type RAS proteins in determining overall RAS-ERK pathway activity. Together, these two advances comprise a new opportunity for therapeutic intervention. In this review, we evaluate miR-based therapeutic strategies for modulating RAS-ERK signaling in cancers, in particular for more direct modulation of RAS-GTP levels, with the potential to complement current strategies in order to yield more durable treatment responses. To this end, we discuss the potential for miR-based therapies focused on three prominent miRs including the pan-RAS regulator let-7 and the GAP regulator comprised of miR-206 and miR-21 (miR-206/21).

INTRODUCTION

MicroRNAs (miRs) are genomically encoded single stranded noncoding RNAs that are typically 19–25 nucleotides (nt) in length and result from extensive processing of endogenous hairpin-shaped precursors [He and Hannon, 2004; Kim, 2005; Chen and Rajewsky, 2007; Ameres and Zamore, 2013]. miRs were initially identified in C. elegans as gene products required for the regulation of proper developmental timing [Wightman et al., 1991; Lee et al., 1993]. Subsequently, thousands of miRs and putative miR-encoding genes have been identified in a wide variety of organisms, including in plants and metazoans. Since their discovery, miRs have emerged as critical regulators of gene expression and cell signaling, and are functionally implicated in numerous cellular processes including development, differentiation, proliferation, and apoptosis [Kasinski and Slack, 2011; Sayed and Abdellatif, 2011; Iorio and Croce, 2012; Sun and Lai, 2013]. As a consequence of these critical roles, dysregulation of miR expression, activity, and signaling results in a multitude of pathological states, including the development and progression of cancers [Esquela-Kerscher and Slack, 2006; Calin and Croce, 2006a; Calin and Croce, 2006b].

Perturbation of key signaling networks endows cells with many of the well-established hallmarks of cancer, such as enhanced cell proliferation, resistance to cell stress and death, and motility, and is implicated in the pathogenesis of virtually every type of human malignancy [Hanahan and Weinberg, 2000; Weinberg, 2007]. Attributed to elevated levels of membrane-associated RAS-GTP, signaling downstream of RAS proto-oncogenes through the RAF-MEK-ERK mitogen activated protein kinase (MAPK) pathway is often inappropriately activated in a wide variety of cancers, promoting several of the classical hallmarks of cancer [Bos, 1989; Schubbert et al., 2007; Bos et al., 2007; Karnoub and Weinberg, 2008; Young et al., 2009; Tidyman and Rauen, 2009; Pylayeva-Gupta et al., 2011]. Activation of this hierarchically tiered signaling pathway can occur through a variety of ways, including in response to stimulation by upstream inputs (i.e., receptor tyrosine kinases (RTKs), integrins, ion channels, etc.), somatic mutation of pathway components such as RAS and RAF, and alteration of the expression of pathway regulators [Schlessinger, 2000; Johnson and Lapadat, 2002; Downward, 2003; Kolch, 2005; Dhillon et al., 2007; Mebratu and Tesfaigzi, 2009]. In cancer cells, the activation of RAS-ERK signaling has been most prominently documented in the context of somatic acquisition of activating point mutations in RAS GTPase genes (e.g., KRAS, HRAS, NRAS). These mutations render the encoded gene products resistant to the inhibitory action of GTPase activating proteins (GAPs, which potently stimulate GTP hydrolysis by RAS) [Trahey and Mccormick, 1987; Boguski and Mccormick, 1993; Scheffzek et al., 1995; Scheffzek et al., 1997; Bos et al., 2007]. In addition to GAPs, numerous factors contribute to the proper spatio-temporal regulation of RAS-ERK signaling, including guanine nucleotide exchange factors (GEFs), which promote recycling to the active, GTP bound state by reducing the affinity of RAS proteins for GDP. In addition, other proteins function as scaffolds or adaptors for the proper localization of signaling molecules, such as SPRED1 which is critical for the membrane localization of NF1/GAP [Bos et al., 2007; McKay and Morrison, 2007; Wortzel and Seger, 2011; Stowe et al., 2012; Roskoski, Jr., 2012a; Roskoski, Jr., 2012b]. More recently, analyses of tumor cells containing a RAS mutation indicated that the wild type proteins encoded by the remaining, unmutated RAS alleles play a critical role in pathway output, identifying these wild type proteins as a potential Achilles’ heel for therapeutic targeting [Jeng et al., 2012; Young et al., 2013; Grabocka et al., 2014; Sharma et al., 2014]

miRs represent yet another level of regulatory control of RAS-ERK signaling and, in certain tumor cells such as basal-like or triple-negative breast cancer (TNBC), can represent major regulators of RAS-ERK activity by impacting the translation of pathway components such as GAPs and/or GAP-associated scaffolding proteins such as SPRED1 [Johnson et al., 2005; Fish et al., 2008; Thum et al., 2008; Hatley et al., 2010; Sun et al., 2013; Sharma et al., 2014; Stark et al., 2015b]. Unraveling how miRs impact RAS-ERK signaling in cancer has the potential to uncover novel therapeutic strategies which can complement conventional modalities and/or targeted therapies such as kinase inhibitors. In this review, we briefly describe miR biogenesis and how miRs can impact the pathogenesis of cancer by altering cell signaling. We discuss miR-based therapeutic strategies and necessary considerations for the successful use of in vivo miR-targeting agents. We then describe the structure of the circuitry of the RAS-ERK signaling pathway, and briefly review the utility of inhibiting this pathway in the treatment of cancers. We consider how miRs can regulate RAS-ERK signaling by targeting specific pathway components and critical regulatory proteins, including wild-type RAS proteins. We next evaluate the prospect of targeting miR-mediated regulation of RAS-ERK in the therapy of cancers and contrast this therapeutic modality with other pharmacological RAS-ERK inhibitory strategies. We conclude by proposing a therapeutic strategy for the more direct suppression of RAS-GTP levels, including the in vivo silencing of the cooperative GAP regulators, miR-206 and miR-21 (i.e., miR-206/21). Recent studies indicate that these two miRs maintain RAS-ERK signaling in breast cancer cells by limiting the translation of a major GAP termed RASA1 and the NF1/GAP-associated factor, SPRED1 [Sharma et al., 2014]. In TNBC cells the resulting suppression of GAP activities by endogenous miR-206/21 is critical for the maintenance of wild type RAS-GTP levels, RAS-ERK signaling and malignant properties not only for cancer cells harboring wild type RAS proteins, but similarly in tumor cells harboring a RAS mutation, further supporting a critical role of wild type RAS in cells harboring the mutant protein [Jeng et al., 2012; Young et al., 2013; Grabocka et al., 2014; Sharma et al., 2014].

MICRORNAS

miR Biogenesis

In humans, the predominant miR biosynthetic route involves the transcription of miR-encoding genes by RNA polymerase II [He and Hannon, 2004; Kim, 2005; Chen and Rajewsky, 2007; Ameres and Zamore, 2013]. The resulting primary miR transcripts (pri-miRs) are processed in the nucleus by the RNAse-III enzyme Drosha, which exists as part of a heterodimeric complex with DGCR8 microprocessor complex unit (also known as “DiGeorge Syndrome Critical Region 8”) to yield stem-loop hairpin structures of approximately 70 nt in length, termed precursor miRs (pre-miRs) [Lee et al., 2003; Denli et al., 2004; Gregory et al., 2004; Landthaler et al., 2004; Han et al., 2004]. Pre-miRs are exported out of the nucleus predominantly by the action of Ran-GTPase/exportin-5 and further processed by the cytoplasmic RNAse-III enzyme Dicer, to yield RNA duplexes composed of the mature miRs of approximately 19–25 nt in length [Bernstein et al., 2001; Ketting et al., 2001; Provost et al., 2002; Lee et al., 2002; Lee et al., 2003]. These are termed miR-miR* duplexes. Following strand selection, “miR” represents the more abundant strand of the duplex and the less abundant strand is denoted “miR*” [Ambros et al., 2003; Griffiths-Jones et al., 2006]. In addition to this major route of biogenesis, some miRs arise from alternate synthetic pathways, including processes that do not utilize RNA polymerase II mediated transcription, or the action of Drosha/DGCR8 or Dicer proteins [Yang and Lai, 2011]. For example, miR-genes located near Alu-repeat sequences or tRNA genes can be transcribed by RNA polymerase III [Borchert et al., 2006]. Furthermore, miRs can also arise from a Drosha/DGCR8 independent synthetic process from the splicing of intronic regions from mRNA transcripts and are termed miRtrons [Okamura et al., 2007]. Finally, miR-miR* duplexes may be produced by direct cleavage of RNA precursors by the endoribonuclease Argonaute-2 (AGO2) to yield mature miRs in a Dicer independent process [Yang et al., 2010].

miR-mediated Regulation of Protein Translation

Mature miRs typically repress the translation of mRNA transcripts by associating with the catalytic center (AGO endoribonucleases) of multiprotein complexes termed RNA-induced silencing complexes (RISCs) [Bartel, 2009; Czech and Hannon, 2011]. miR-miR* duplexes are subsequently unwound and a mature miR strand is retained in RISC based on the relative thermodynamic stability across the miR-miR* duplex. The miR* strand may subsequently either be cleaved or be ejected from the assembled complex. The mature RISC then scans target mRNA sequences. The selectivity for target mRNAs arises from miR sequence complementarity to portions of the target mRNAs, particularly in the 3′ untranslated region (3′ UTR). In mammals, sequence complementarity between bases 2–8 near the 5′ end of the miR (termed the miR-seed sequence) and portions of the mRNA is the dominant factor that guides RISCs to target and to repress the translation of specific transcripts [He and Hannon, 2004; Chen and Rajewsky, 2007; Jackson et al., 2010; Ameres and Zamore, 2013]. The resulting miR-mediated translational repression may occur by (i) blocking translation initiation, (ii) enhanced mRNA degradation, or by (iii) site specific cleavage of the target mRNA, though the latter process occurs infrequently in mammals [He and Hannon, 2004; Chen and Rajewsky, 2007; Jackson et al., 2010; Ameres and Zamore, 2013]. Interestingly, though miRs typically repress the translation of target mRNAs, a few miRs have been documented to promote the translation of cognate transcripts, suggesting an additional level of complexity in this modality of regulating gene expression [Vasudevan and Steitz, 2007; Vasudevan et al., 2007; Vasudevan et al., 2008; Steitz and Vasudevan, 2009; Jangra et al., 2010; Lin et al., 2011; Ameres and Zamore, 2013].

miRs are capable of widespread regulation of gene expression, with more than 60% of protein coding mRNA transcripts possessing at least one evolutionarily conserved miR seed complementary sequence [Ha and Kim, 2014]. Conversely, whereas the translation of a single mRNA transcript may be impacted by multiple miRs, individual miRs can potentially regulate the expression of hundreds of protein coding transcripts, each of which may have diverse cellular functions. Importantly, the collaborative action of miRs can potently modulate the activity of key signaling networks by targeting one or more pathway components [He and Hannon, 2004; Chen and Rajewsky, 2007; Jackson et al., 2010; Ameres and Zamore, 2013]. miRs may also participate in reciprocal regulation of transcripts that are critical for miR biogenesis or maturation, and thus constitute components of feedback loops along with their target mRNAs.

These complex miR-mRNA interaction paradigms are essential for the maintenance of homeostasis of critical cell physiological processes and the perturbation of miR regulation of important cell signaling networks, such as the RAS-ERK pathway, contributes to a wide variety of pathological states, including cancer. Indeed, recent work suggests that miR signaling to RAS-ERK can be responsible for maintaining low GAP activity that leads to high RAS-ERK pathway activity, regardless of the RAS mutational status [Sharma et al., 2014]. Furthermore, studies indicate that mutant RAS-GTP level does not on its own determine pathway activity, but instead that both the mutant and wild type RAS proteins must preferentially associate with GTP in order to maintain higher levels of pathway activity [Jeng et al., 2012; Young et al., 2013; Grabocka et al., 2014; Sharma et al., 2014].

miR-based Therapeutic Strategies

As miR dysregulation is a prominent feature of many pathological states, the therapeutic targeting of specific miRs holds the promise for therapy of various diseases [Kasinski and Slack, 2010; Inui et al., 2010; Kasinski and Slack, 2011; Stenvang et al., 2012; Ling et al., 2013; Li and Rana, 2014; van and Kauppinen, 2014]. miR-based therapeutics are defined as strategies that restore or inhibit miR function to counteract perturbations in miR-signaling. These strategies include (i) restoring miR function by supplementation of miR-mimics, (ii) inhibiting miR function by synthetic anti-sense oligonucleotide-based approaches targeting endogenous miRs (termed anti-miRs and antagomiRs) or by (iii) modulating miR function by non-oligonucleotide based methods including peptide nucleic acids (PNAs) [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014]. Furthermore, oligonucleotide based strategies may feature various chemical modifications to enhance the stability and affinity of these therapeutic agents [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014].

Despite our extensive knowledge of attractive miR targets in various disease states, the successful utilization of miR-based therapeutic strategies in vivo is challenged by several obstacles [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014]. First, the successful delivery of these agents to the target tissue is limited by physical, anatomical, pharmacokinetic, and pharmacodynamic barriers, which may be overcome in part by a variety of delivery methods and targeting strategies including miR-cholesterol conjugation, liposome encapsulation, miR-nanoparticle conjugation, and antibody- or aptamer-based targeting methods [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014]. As miR-mimics and anti-miRs can be degraded by ubiquitously present nucleases in the human body, synthetic modification of these agents is necessary for their stability [Lamond and Sproat, 1993; Cummins et al., 1995].

Second, miR-based therapies, including anti-sense strategies, have the potential for promiscuous miR inhibition, yielding off-target effects [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014]. miR-therapeutics must be able to discern between the many miRs that may share identical/similar seed sequences with the intended target. Thus, careful consideration of target miRs must be undertaken to ensure that miR-based therapeutics can modulate the intended endogenous target with a high degree of specificity, to minimize off-target effects.

Finally, the administration of miR-therapeutics and carrier vehicles such as targeted nanoparticles may cause deleterious consequences by miR sequence independent off-target effects [Kasinski and Slack, 2011; Ling et al., 2013; Li and Rana, 2014]. miR agents may be detected by both the innate and adaptive arms of the human immune systems, and chemical modification to therapeutic components is necessary to avoid immunostimulatory off-target effects [Judge et al., 2005; Hornung et al., 2005]. Furthermore, these agents may alter physiological processes (e.g., blood coagulation, complement cascade activation) or may induce organ system dysfunction (e.g., hepatotoxicity, nephrotoxicity), owing to the accumulation, clearance, and excretion of these molecules [Galbraith et al., 1994; Henry et al., 1997; Swayze et al., 2007]. Therefore, the consideration of these therapeutic barriers is necessary for the successful utilization of miR-based therapeutics.

RAS-ERK SIGNALING

Organization and Regulation of the RAS-ERK Signaling Pathway

RAS-ERK signaling is a critical mediator of cell physiological processes including cell proliferation, differentiation and motility [Johnson and Lapadat, 2002; Downward, 2003; Kolch, 2005; Dhillon et al., 2007; Young et al., 2009; Mebratu and Tesfaigzi, 2009; Pylayeva-Gupta et al., 2011]. Activation of this pathway occurs downstream of signaling inputs including receptor tyrosine kinases (RTKs), integrins, and ion channels, which are in turn activated by a variety of stimuli and cell stresses [Schlessinger, 2000; Johnson and Lapadat, 2002; Lemmon and Schlessinger, 2010]. The RAS family of GTPases consists of four members (HRAS, NRAS, KRAS4A and KRAS4B [alternatively spliced variants]) that arise from three distinct genes (HRAS, NRAS, and KRAS) and occupy a critical position in relaying signaling from these diverse inputs to activate downstream effector pathways such as the RAF-MEK-ERK pathway, as well as the phosphoinositide 3-kinase (PI3-K)-AKT pathway [McCubrey et al., 2006; Roberts and Der, 2007; Young et al., 2009]. RAS proteins fulfill this important role by functioning as binary switches that alternate between the GTP-bound “on” state (RAS-GTP), which enables RAS to engage downstream effector pathways, and the GDP-bound “off” state (RAS-GDP) [Bos et al., 2007; Vigil et al., 2010]. The activation state of RAS is predominantly governed by critical accessory proteins that enable the transition between either of these states. Essential factors for proper RAS signaling include guanine nucleotide exchange factors (GEFs) which promote the formation of RAS-GTP, GAPs, which promote GTP hydrolysis, and scaffolding proteins such as SPRED1 which appears critical for membrane localization of NF1/GAP [Stowe et al., 2012].

In humans, the activation of the RAF-MEK-ERK pathway is initiated by the preferential interaction of membrane-associated RAS-GTP with the RAS-binding domain of the RAF family of serine/threonine kinases [Karnoub and Weinberg, 2008; Cox and Der, 2010; Pylayeva-Gupta et al., 2011]. Membrane-associated RAS proteins exist as dimers, and this dimerization may be critical for the activation of RAF kinases (composed of three paralogs: ARAF, BRAF, and CRAF/RAF-1), which occurs in a complex multi-step process [Inouye et al., 2000; Guldenhaupt et al., 2012; Lin et al., 2014]. Activated RAF kinases phosphorylate and activate the dual specificity kinases MEK 1 and MEK 2 (MEK 1/2), which in turn phosphorylate and activate ERK 1 and ERK 2 (ERK 1/2), the terminal effector kinases of this pathway [Johnson and Lapadat, 2002; Roskoski, Jr., 2012a; Roskoski, Jr., 2012b]. In contrast to the limited substrate specificity of RAF and MEK 1/2, ERK 1/2 are capable of phosphorylating and consequently modulating the activity of a wide variety of cytoplasmic and nuclear substrates. Importantly, the activity of ERK 1/2 responsive transcription factors is critical in orchestrating cell responses to numerous input stimuli that lie upstream of RAS GTPases [Roskoski, Jr., 2012a; Roskoski, Jr., 2012b].

Regulation of RAS-ERK Pathway Activity

The activation of RAS-ERK signaling is tightly regulated through a variety of means. RAS-ERK pathway activity is maintained by a delicate balance between factors that promote pathway activation (i.e., GEFs), factors that inhibit pathway activation (i.e., GAPs, DUSPs), and proteins that function as scaffolds, adaptors, and/or provide docking sites for pathway regulatory components [Boguski and Mccormick, 1993; Bos et al., 2007; McKay and Morrison, 2007; Yoshimura, 2009; Cox and Der, 2010; Vigil et al., 2010; Wortzel and Seger, 2011; Roskoski, Jr., 2012a; Roskoski, Jr., 2012b]. These factors may confer signaling specificity to membrane subdomains, allowing distinct effects of the different RAS family members. Additionally, ERK 1/2 can directly phosphorylate and inhibit the activity of the GEF SOS1, CRAF, and MEK1, and thus attenuate signaling by feedback inhibition [Ueki et al., 1994; Rossomando et al., 1994; Buday et al., 1995]. Furthermore, ERK 1/2 can also regulate the transcription of upstream drivers of RAS-ERK signaling such as RTKs [Sears et al., 2000; Amit et al., 2007; Lemmon and Schlessinger, 2010; Duncan et al., 2012].

Tightly controlled spatio-temporal regulation of RAS-ERK signaling is critical for the proper execution of cell physiological processes, and inappropriate regulation of the pathway results in a variety of disease states, including developmental disorders and cancer [Bos et al., 2007; McKay and Morrison, 2007; Wortzel and Seger, 2011; Roskoski, Jr., 2012a; Roskoski, Jr., 2012b]. For example, somatic activating point mutation of RAS and BRAF genes occur in approximately 15–30% and 7–8% of all cancers respectively [Davies et al., 2002; Downward, 2003; Schubbert et al., 2007; Karnoub and Weinberg, 2008; Young et al., 2009; Tidyman and Rauen, 2009; Cox and Der, 2010; Pylayeva-Gupta et al., 2011; Fernandez-Medarde and Santos, 2011]. Though some malignancies, such as pancreatic ductal adenocarcinomas, colorectal carcinomas, and melanomas feature a high proportion of activating RAS and RAF mutations [Bos, 1989; Pylayeva-Gupta et al., 2011], other cancers such as TNBCs display RAS-ERK pathway activation despite the infrequent occurrence of somatic point mutations, thus implicating dysregulation of RAS-ERK through other means [Mirzoeva et al., 2009; Hoeflich et al., 2009b; Cancer Genome, 2012]. Interestingly, cancers such as TNBCs frequently display genetic alterations such as gene copy number changes in pathway components and altered expression of pathway regulatory proteins [van Beers et al., 2005; Herschkowitz et al., 2007; Rakha et al., 2008; Hu et al., 2009; Cancer Genome, 2012; Balko et al., 2012; Balko et al., 2013]. Remarkably, 32% of basal-like breast cancers display KRAS gene amplifications and 30% of cancers of this subtype harbor BRAF gene amplifications [Cancer Genome, 2012].

The Emerging Paradigm of a Critical Role for Wild Type RAS Proteins in Cells Harboring RAS Mutations

Early seminal studies identified the potent transforming ability of virally encoded RAS genes and subsequently characterized these gene products as mutated versions of the human RAS homologs [Harvey, 1964; Kirsten and Mayer, 1967; Scolnick et al., 1973; Scolnick and Parks, 1974; Shih et al., 1979; Krontiris and Cooper, 1981; Santos et al., 1982; Chang et al., 1982; Parada et al., 1982; Der et al., 1982]. Furthermore, the observation of similarly mutated endogenous RAS genes in human tumor samples was critical for our understanding of the molecular basis of carcinogenesis [Capon et al., 1983; Feig et al., 1984; Santos et al., 1984]. These important early studies uncovered that RAS mutations predominantly occur in codons 12, 13, and 61 and that these mutant proteins were constitutively bound to GTP [Taparowsky et al., 1982; Reddy et al., 1982; Tabin et al., 1982; Hall et al., 1983]. Furthermore, mutant RAS possessed far less intrinsic GTPase activity compared to the non-mutant counterparts and were virtually resistant to the action of GAPs [Sweet et al., 1984; Gibbs et al., 1984; McGrath et al., 1984; Manne et al., 1985; Trahey and Mccormick, 1987; Mccormick et al., 1991; Mccormick, 1992]. The striking effect of activated RAS in these early experiments and the identification of RAS mutations in cancers sparked several decades of research that has vastly broadened our knowledge of cell signaling and its role in neoplasia.

But compelling questions regarding the regulation of RAS signaling still remain. Recently, how the activity of wild-type RAS proteins contributes to downstream pathway activation in the context of RAS-mutant cells was uncovered in a series of genetic and biochemical studies [Jeng et al., 2012; Young et al., 2013; Grabocka et al., 2014], including a study by our lab focused on the role of miR-regulated GAPs in RAS-mutant cells [Sharma et al., 2014]. These studies uncovered a previously unappreciated role for wild-type RAS proteins, as well as GEFs and GAPs, as critical signaling molecules in the context of mutant-RAS associated phenotypes. Possibly attributed to the formation of wild type/mutant RAS dimers, these studies found that GEFs and GAPs are critical regulators of tumorigenesis of RAS-mutant cells through their modulation of WT-RAS-GTP levels. These studies concluded that regardless of the RAS mutational status, the ultimate signaling output is likely determined by the ratio of RAS-GTP to RAS-GDP, where the pool of WT-RAS plays a major role, even in cells with mutant RAS. A critical difference between wild-type and mutant RAS proteins is the greater dependence of WT-RAS on GEFs and GAPs [Bos et al., 2007; Vigil et al., 2010]. Consequently these new studies identify these regulators as critical therapeutic targets regardless of the RAS mutational status. These studies establish a new paradigm of how RAS signaling is regulated and highlight the potential of small molecule modulators of GAP or GEF activity.

Therapeutic Targeting of RAS-ERK Signaling in Cancers

Given the important role of RAS-ERK signaling in the development and progression of many cancers, the successful therapeutic inhibition of this pathway has been a long standing goal of the targeted chemotherapy era [Downward, 2003; Karnoub and Weinberg, 2008; Gysin et al., 2011; Pylayeva-Gupta et al., 2011; Mattingly, 2013]. Numerous strategies to inhibit RAS-ERK signaling have been envisioned, including those that (i) directly target RAS, (ii) modulate factors that regulate RAS activity, and those that (iii) target downstream kinases (e.g. RAF, MEK, and ERK). One of the most promising therapeutic strategies, utilizing ATP analogues as allosteric or competitive inhibitors of RAF or MEK kinase activity has proceeded toward clinical utility, but with prolonged therapeutic responses limited by a variety of factors [Liu et al., 2013; Johnson et al., 2014]. Despite the potent action of these compounds in vitro, therapeutic resistance emerges rapidly and hampers the successful use of these kinase inhibitors [Engelman et al., 2007; Garrett et al., 2011; Chandarlapaty et al., 2011; Duncan et al., 2012; Johnson et al., 2014; Stuhlmiller et al., 2015].

Contributing to this resistance, acute loss of RAS-ERK signaling in cancer cells results in adaptive reprogramming, including reprogramming of the kinome, with upregulation of multiple (receptor tyrosine) kinases (RTKs) including PDGFRβ, DDR1, and others [Engelman et al., 2007; Garrett et al., 2011; Chandarlapaty et al., 2011; Duncan et al., 2012; Johnson et al., 2014; Stuhlmiller et al., 2015]. In addition, pathway inhibition by agents such as MEK inhibitor is thwarted by the loss of negative feedback regulation, including ERK 1/2 mediated inhibition of positive pathway regulators such as MEK1 and BRAF [Duncan et al., 2012; Johnson et al., 2014]. Another factor is that phosphorylation of MEK1 by cRAF results in reduced affinity of MEK 1/2 allosteric inhibitors [Ueki et al., 1994; Ritt et al., 2010]. Furthermore, and particularly in melanoma, long term treatment with RAS-ERK inhibitory compounds results in tumor cells acquiring somatic mutations in NRAS, MEK2, or AKT1 to counteract sustained inhibited signaling [Nazarian et al., 2010; Villanueva et al., 2013; Shi et al., 2014]. Finally, through the induction of multiple RTKs, the activation of alternative signaling pathways (e.g., PI3-K-AKT) can compensate for the inhibited RAS-ERK signaling [Sos et al., 2009; Hoeflich et al., 2009a; Nazarian et al., 2010; Duncan et al., 2012; Sun et al., 2014]. Either singly or in combination, these adaptive changes ultimately circumvent blocked signaling and prevent sustained therapeutic responses. Thus, the development and optimization of more effective RAS-ERK pathway inhibitory strategies and counteracting the rapid emergence of resistant signaling represent critical obstacles to effective therapeutic intervention.

A major goal has been more direct methods for suppression of RAS-GTP levels [Gysin et al., 2011; Mattingly, 2013]. Toward this end, the recent recognition that WT RAS proteins play a critical role in pathway output would appear to reenergize ongoing efforts to target GEFs and GAPs [Bos et al., 2007; Vigil et al., 2010]. Other ongoing approaches include reovirus-based therapies and siRNA therapy against mutant KRAS [Thirukkumaran and Morris, 2009; Zorde et al., 2013; Yuan et al., 2014; Golan et al., 2015].

MICRORNA REGULATION OF MUTANT AND WILD TYPE RAS-GTP

The past decade has provided considerable insight into the critical regulatory roles that miRs exert over key cancer relevant signaling networks such as the RAS-ERK pathway. Our knowledge of how miRs can modulate RAS-ERK pathway activation continues to grow as potential miR-mRNA regulatory networks are identified using a variety of strategies including in silico miR target prediction methods, profiling of the cellular transcriptome/proteome, and experimental validation of putative interactors that typically employs translational reporter assays.

Three major paradigms of miR-mediated RAS-ERK regulation have emerged from these studies. miRs can impact the translation of (i) core RAS-ERK pathway components (e.g., let-7 targets HRAS, NRAS, and KRAS) [Johnson et al., 2005], (ii) critical pathway regulatory proteins that are required for the proper spatio-temporal control of RAS-ERK signaling (e.g., miR-206 and/or miR-21 collaboratively target RASA1, SPRED1, SPRY1; and miR-21 individually targets PTEN) (Fig. 1) [Meng et al., 2007; Hatley et al., 2010; Sharma et al., 2014], and (iii) upstream drivers and downstream effector/regulatory molecules (e.g., miR-9-3p targets ITGB1, and miR-206/21 co-target PDCD4) [Zawistowski et al., 2013; Lin et al., 2015]. Examples of miRs that regulate RAS-ERK pathway activity in a variety of cancer contexts are listed in Table 1. Indeed, these miRs represent potential therapeutic substrates and targets that can be modulated in the treatment of cancer. In the following sections, we evaluate the therapeutic potential of three miRs, let-7 and miR-206/21, that hold great promise as potential therapeutic targets in the treatment of cancers by impacting RAS-ERK signaling.

Figure 1. miRs regulate RAS-ERK pathway activity by regulation of RAS-GTP.

The schematic shows the organization of the RAS-ERK pathway. miR-206/21 co-targeted repressors of RAS-ERK signaling are indicated in ovals. The GAP protein NF1 is indicated as a likely catalytic partner of SPRED1 [Stowe et al., 2012]. The let-7 miR targets each of the RAS family GTPases including KRAS, HRAS, and NRAS.

Table 1.

miRs that regulate RAS-ERK pathway activity in a variety of cancer contexts

miR	Target(s)	Disease contexts	References
let-7	HRAS, NRAS, KRAS, cMYC	Multiple cancer contexts including lung adenocarcinoma	[Johnson et al., 2005] [Johnson et al., 2007] [Yu et al., 2007] [Kasinski and Slack, 2010] [He et al., 2010]
miR-21	RASA1, SPRED1, SPRY1, SPRY2, PTEN, PDCD4	Multiple cancer contexts including TNBCs	[Iorio et al., 2005] [Volinia et al., 2006] [Frankel et al., 2008] [Jin et al., 2013] [Mei et al., 2013] [Wickramasinghe et al., 2009] [Asangani et al., 2008] [Meng et al., 2006] [Sharma et al., 2014]
miR-206	RASA1, SPRED1, PDCD4	TNBCs	[Sharma et al., 2014] [Lin et al., 2015]
miR-31	RASA1	Colorectal carcinoma	[Sun et al., 2013]
miR-143	NRAS	Glioma	[Wang et al., 2014]
miR-514a	NF1	Melanoma	[Stark et al., 2015a]
miR-223	RASA1	Colorectal adenocarcinoma	[Sun et al., 2015]
miR-181a	KRAS	Oral squamous cell carcinoma	[Shin et al., 2011]
miR-524-5p	BRAF, ERK2	Melanoma	[Liu et al., 2014]
miR-96	KRAS	Pancreatic adenocarcinoma	[Yu et al., 2010]
miR-30c	KRAS	Breast cancer	[Tanic et al., 2012]

Let-7 Represses the Translation of the RAS Family of GTPases

The let-7 gene was initially identified as an essential regulator of patterning development in the nematode C. elegans, and was among the first defined miRs [Wightman et al., 1991; Lee et al., 1993]. Subsequent studies observed evolutionary conservation of let-7 and identified related paralogs in the genomes of multiple species, including humans [Pasquinelli et al., 2000; Reinhart et al., 2000]. Similarly to C. elegans, human let-7 is critical for epithelial cellular differentiation and proliferation [Yu et al., 2007; Johnson et al., 2007]. Furthermore, reduced expression of let-7 in cancer occurs through genetic deletion, mutation, epigenetic silencing, and post-transcriptional regulation of let-7 biogenesis, and decreased let-7 expression has been implicated in pathogenesis of a wide variety of malignancies, including cancers of the lung, colon, ovary, and breast [Johnson et al., 2005; Yu et al., 2007; Kasinski and Slack, 2010]. These studies suggest a tumor suppressive function for this miR.

Prominent mechanisms by which let-7 exerts a tumor suppressive role is by repressing the translation of the three RAS proteins (HRAS, NRAS, and KRAS) and cMYC, a downstream effector of RAS-ERK signaling [Johnson et al., 2005; He et al., 2010]. Studies analyzing in vitro and in vivo models of non-small cell lung cancer (NSCLC), as well as human tumor samples, show that let-7 expression is inversely correlated with the expression of KRAS, a critical promoter of NSCLC tumorigenesis. Let-7 abrogates tumor development and RAS-ERK signaling in an autochthonous model of NSCLC driven by activated KRAS (KRAS^G12D) [Esquela-Kerscher et al., 2008; Kumar et al., 2008]. Consistent with this previous result, a tumor suppressive role for let-7 was observed in a study analyzing a xenograft model of NSCLC [He et al., 2010]. Additionally, in a breast cancer context, let-7 antagonizes the maintenance, survival, and self-renewal of cancer stem-like cells (CSCs), and this suppressive activity was correlated with the reduced expression of RAS and HMGA2 [Yu et al., 2007]. Thus, by suppressing RAS expression, let-7 can attenuate RAF-MEK-ERK signaling and dependent oncogenic phenotypes regardless of the RAS-mutation status of cancers. These studies suggest that let-7 can act as both a cancer-preventative and cancer-therapeutic agent, and point to let-7 supplementation as a promising strategy to target RAS-ERK signaling in the treatment of cancers.

MiR-206/21 Collaboratively Repress the Translation of RASA1 and SPRED1 and Inhibit GAP Activity

Our laboratory recently identified two miRs, miR-206 and the well characterized oncogene miR-21 (collectively: miR-206/21), as critical regulators of RAS-ERK signaling in TNBC cells (Fig. 1) [Sharma et al., 2014]. Whereas miR-206 is well characterized in regulating the differentiation of adult muscle stem cells, the role of endogenous miR-206 in breast cancer is less well known [Kim et al., 2006; Chen et al., 2010; Dey et al., 2011; Liu et al., 2012]. In contrast to miR-206, miR-21 is prominently upregulated in many malignancies, including in breast cancer, and promotes tumorigenesis by repressing the translation of multiple tumor suppressors, including negative regulators of RAS-ERK signaling (e.g., SPRY1, RASA1, and PDCD4), as well as RAS-PI3K signaling (e.g., PTEN) [Iorio et al., 2005; Volinia et al., 2006; Meng et al., 2006; Frankel et al., 2008; Thum et al., 2008; Asangani et al., 2008; Wickramasinghe et al., 2009; Jin et al., 2013; Mei et al., 2013].

We found that the expression of miR-206/21 was dependent on the zinc-finger pluripotency factor Kruppel-like factor 4 (KLF4), which has most often been implicated as a poor prognostic factor in breast cancer [Pandya et al., 2004; Kamalakaran et al., 2011; Chen et al., 2012]. Whereas KLF4 and miR-206 are preferentially expressed in MaCSCs, miR-21 is similarly expressed in these two compartments, consistent with the “on/off” mode of miR-21 regulation by KLF4 [Sharma et al., 2014; Lin et al., 2015]. Furthermore, recent studies from our laboratory indicate that KLF4 and/or its dependent miRs are important regulators of anti-apoptotic signaling in breast cancer cells and promote survival against diverse forms of stress, including treatment with conventional cytotoxic or targeted chemotherapies [Farrugia et al., 2015; Lin et al., 2015].

The combined action of miR-206/21 promotes signaling by repressing the translation of multiple RAS-ERK pathway inhibitory proteins which act at various hierarchical levels in this signaling network [Sharma et al., 2014]. Interestingly, the manipulation of each individual miR did not yield large changes in pathway activity, suggesting that the collaborative action of miR-206/21 was required to achieve a substantial effect. Indeed, treatment of TNBC cells with anti-miR-206/21 was sufficient to suppress pathway activity by greater than 80%. We found that miR-206/21 co-target and co-suppress the translation of the GAP RASA1, and the Neurofibromatosis 1 (NF1) GAP associated protein, SPRED1. This GAP-deficient state interferes with RAS inactivation (i.e., the formation of signaling-deficient WT-RAS-GDP), and consequently promotes RAS-ERK signaling, RAS dependent cell phenotypes, and TNBC tumorigenesis. Importantly, whereas inhibition of KLF4-miR-206/21 signaling potently suppresses RAS-ERK signaling in multiple RAS-mutant TNBC models (MDA-MB-231– KRAS^G13D, Hs578t – HRAS^G12D, and SUM159PT – HRAS^G12D) as well as in cells that exclusively harbor WT-RAS proteins, stable shRNA-mediated suppression of RASA1 and SPRED1 promoted pathway activity on its own, and rendered cells virtually resistant to anti-miR-206/21. Consequently, TNBC cells and potentially many other tumor types are GAP-deficient owing to the collaborative action of miR-206/21 on RASA1 and SPRED1. Therefore the RASA1 and SPRED1 transcripts represent latent tumor suppressors with the potential for reactivation by anti-miR-206/21. Our analysis of GAP signaling and RAS-GTP levels in this study yielded results that were consistent with the newly emerging paradigm that the output of RAS-ERK signaling is critically dependent on the activation status of WT-RAS, and uncovered a previously unappreciated role for GAP proteins in cells harboring RAS mutations [Jeng et al., 2012; Young et al., 2013; Grabocka et al., 2014; Sharma et al., 2014].

The Potential for in vivo Inhibition of MiR-206/21 for the Treatment of Cancer

In addition to the numerous studies that have elucidated the oncogenic role of miR-21, our analysis of KLF4-miR signaling suggests that inhibition of miR-206/21 has the potential to be a promising therapeutic strategy in the treatment of TNBC and in other cancers. In vivo silencing of miR-206/21 using anti-sense oligonucleotide based strategies could effectively attenuate RAS-ERK signaling by more directly suppressing RAS-GTP levels. Improved therapeutic effects may be achieved when used in combination with other pathway inhibitory strategies or in conjunction with cytotoxic chemotherapy. Furthermore, other strategies for suppression of oncogenic miRs hold promise, including aptamer-mediated inhibition of nucleolin [Pichiorri et al., 2013], a cell surface protein required for the maturation of a specific subset of miRs, including miRs that promote RAS-ERK signaling (e.g., miR-21, miR-221).

Therapeutic targeting of these miRs in combination with other therapeutic modalities could offer an advantage over single kinase inhibition strategies. Due to the rapid adaptive reprogramming and the emergence of inhibitor-resistant signaling, these do not yield durable responses [Engelman et al., 2007; Garrett et al., 2011; Chandarlapaty et al., 2011; Duncan et al., 2012; Johnson et al., 2014; Stuhlmiller et al., 2015]. Probably owing to the more physiologic enhancement of GAP activity, suppression of miR-206/21 leads to potent inhibition of RAS-ERK signaling and RAS-dependent phenotypes [Sharma et al., 2014], but appears not to destabilize c-MYC or to induce the dramatic adaptive reprogramming response that results from kinase inhibitors (unpublished observations, SBS and JMR). Additionally, miR-206/21 suppression may sensitize cells to the effects of other anti-cancer therapeutic modalities. We have recently found that miR-206 represses the translation of the pro-apoptotic protein PDCD4, a well established miR-21 target, to protect breast cancer cells from apoptosis in response to cytotoxic chemotherapy [Lin et al., 2015]. Thus the combinatorial suppression of miR-206/21 could potentiate the effects of conventional treatments and thereby reduce the required dosage of these agents, potentially mitigating the adverse events associated with anti-cancer therapy.

While therapeutic windows are notoriously difficult to predict from the analysis of models, mice deficient in either miR-206 or miR-21 develop normally and appear healthy as adults, supporting the potential for dual inhibition as a therapeutic strategy. Thus, the therapeutic inhibition of miR-206/21 activity has the potential to target RAS-ERK signaling through the re-expression of GAP activity, and holds great promise for the treatment of RAS-driven cancers such as TNBC.

CONCLUSIONS

The RAS-ERK signaling pathway is critical in the development and progression of numerous malignancies, and more effective targeted pathway inhibition has the potential to greatly improve the treatment of cancers. Despite extensive work that has culminated in the development of numerous pathway inhibitory strategies, direct suppression of RAS-GTP levels has been difficult to achieve, and the successful utilization of existing kinase inhibitory agents is hampered by adaptive reprogramming of cell signaling. The consistent demonstration of the inefficacy of single kinase inhibition strategies has prompted the consideration of alternative routes of targeting RAS-ERK signaling components and pathway regulatory molecules. miRs represent one such set of important regulatory molecules that can be targeted for the therapeutic inhibition of pathway activity. These miR-based therapeutic strategies involve the supplementation of RAS-ERK inhibitory miRs using miR-mimics (e.g., let-7), or the in vivo silencing of miRs that promote pathway activity using anti-sense strategies (e.g., miR-206/21). Unlike single kinase inhibitors, these targeted miR-based therapeutic strategies may yield durable anti-tumor responses as they can collaboratively target multiple levels of this signaling pathway and regulate other cell physiologic processes that are critical to RAS-ERK mediated tumorigenesis. Though development and optimization of improved miR delivery methods is necessary, targeting RAS-ERK signaling by miR-based therapeutics holds great promise in the treatment of cancers that are reliant on this signaling pathway.