MicroRNAs as a drug resistance mechanism to targeted therapies in EGFR-mutated NSCLC: Current implications and future directions
Introduction
Lung cancer is the second most common malignancy constituting the leading cause of cancer-related deaths worldwide, accounting for >25% of all cancer deaths (Siegel et al., 2018). Even though lung cancer incidence rates are decreasing in the last years, mainly because of changes in smoking habits, the outcome of lung cancer patients remains dismal (Siegel et al., 2018). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, whereas small-cell lung cancers, which embody a distinct biological entity, constitute the remaining cases (Howlader et al., 2016). Concerning NSCLC, enormous improvements in diagnostic and therapeutic approaches have been made in the past decades. The deeper understanding of NSCLC biology and identification of specific driver mutations have led to the development of targeted treatments that supplanted standard platinum-based chemotherapy in the subset of so-called ‘oncogene-addicted’ NSCLC patients (Network et al., 2014; Ding et al., 2008). The mutations of the Epidermal Growth Factor Receptor (EGFR) gene represent the most common class of targetable genetic aberrations; they can be found in approximately 10–16% of NSCLC patients from Western countries, and this percentage is even higher (up to 50%) in Asian patients (Rosell et al., 2009; Shi et al., 2014). EGFR mutations usually cluster within EGFR exons 18–21, thus impairing the kinase domain of the receptor, that becomes constitutively active, leading to cell proliferation and survival regardless of the presence of the extracellular ligand (Sordella et al., 2004). In-frame deletions in exon 19 and exon 21, and the L858R point mutation are the most frequent mutations (>90%), which confer increased sensitivity to tyrosine kinase inhibitors (TKIs) (Dearden et al., 2013). To date, three generations of TKIs have been developed and tested in the clinical setting: first-generation (gefitinib, erlotinib, and icotinib), second-generation irreversible inhibitors (afatinib and dacomitinib) and highly selective third-generation inhibitors (osimertinib and rociletinib) (Recondo et al., 2018). The introduction of first- and second-generation TKIs for the management of EGFR-driven NSCLC dramatically changed the natural course of the disease. Frontline treatment of patients with these targeted drugs experiences a gain of 3.4–6.9 months in terms of progression-free survival (PFS) over standard chemotherapy (Recondo et al., 2018). However, despite an initial high overall response rate (56–85%), most tumors acquire molecular mechanisms to escape this pathway blocked, resulting in an inexorable progression (Recondo et al., 2018). Over 50% cases of resistance to first- and second-generation TKIs are caused by the onset of the ‘gatekeeper’ mutation T790 M, which compromises the binding of the abovementioned compounds to EGFR and increases the receptor affinity for ATP (Sequist et al., 2011). Third-generation TKI osimertinib can overcome this drug resistance mechanism, and it has proven to be effective in EGFR-mutated NSCLC irrespective of T790 M status. However, the clinical benefit of this drug is limited by the further emergence of drug resistance (Soria et al., 2017). Besides the aforementioned EGFR T790 M mutation leading to detraction of drug binding due to target alteration, other intensively investigated mechanisms of drug resistance in cancer such as overexpression of efflux transporters, increased drug metabolism, epigenetic modifications, DNA damage response and epithelial-to-mesenchymal transition (EMT) were reported (Housman et al., 2014). Another more recently investigated resistance mechanism, lysosomal sequestration, has been described for hydrophobic weak base drugs such as sunitinib and nintedanib (Zhitomirsky and Assaraf, 2016; Gotink et al., 2011; Zhitomirsky and Assaraf, 2017, 2015; Englinger et al., 2017). Focusing on EGFR-mutated NSCLC, secondary EGFR mutations, activation of bypass signaling pathways and histological transformation to small-cell lung cancer constitute peculiar mechanisms that limit the efficacy of EGFR-TKI (Van Der Steen et al., 2018). Thus, an earlier identification of drug resistance mechanisms as well as the development of new strategies to overcome the limitations of EGFR blockade alone, are urgently needed.
Section snippets
An overview of miRNAs
MicroRNAs (miRNAs) are 18–25 nucleotides in length, single-stranded, noncoding RNAs that function as post-transcriptional regulators of gene expression (Krol et al., 2010; Cai et al., 2009). They are synthesized from large precursor RNAs (pri-miRNAs), which are then processed in the nucleus by the RNase III, Drosha, and the double-stranded RNA-binding protein, Pasha, into pre-miRNAs. Pre-miRNAs are then released into the cytoplasm by Exportin 5 and undergo further steps to form the mature
Techniques for miRNAs analysis
Some of the most established molecular biology-based methods, such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and microarrays, remain powerful tools in the quantitative analysis of miRNA. However, these techniques are constantly evolving due to the continuous incorporation of new technologies. RT-PCR methodology to determine miRNAs is based on the role of reverse transcriptase, which converts RNA into their complementary DNA (cDNA) sequences and proceeds to the amplification of
The link between miRNAs and EGFR-mutated NSCLC
The link between miRNAs and lung cancer has been demonstrated by numerous studies over years, as reviewed by Iqbal and collaborators (Iqbal et al., 2018). Cumulative evidence suggests that miRNAs are involved in lung cancer initiation as well as progression. Moreover, miRNAs may be used as biomarkers for the early diagnosis and as predictors of patients’ prognosis (Bjaanæs et al., 2014; Yanaihara et al., 2006; He et al., 2015). For instance, miR-21 has been shown to have a significantly high
Modulation of PI3K/AKT/mTOR signaling pathway
Beyond secondary EGFR mutations, an important mechanism that NSCLC cells can adopt in order to escape EGFR-TKI blockade, relies on the activation of parallel downstream signaling pathways, among which PI3K/AKT/mTOR constitutes a key transduction cascade responsible for cell survival, proliferation and invasion (Vara et al., 2004; Fumarola et al., 2014). PI3K/AKT/mTOR can be activated through the interaction of c-MET, also known as hepatocyte growth factor receptor (HGFR), with its ligand HGF.
Prognostic value
Besides the diagnostic potential of miRNAs in EGFR-mutated NSCLC, the expression of key miRNAs could have prognostic value in the clinical setting. High plasma expression of miR-122, miR-19a, miR-19b, miR-195 and miR-590-5p was associated with better overall survival (OS) among advanced non-smoking female NSCLC patients harboring an EGFR mutation compared to EGFR-wt patients, even after adjusting the results for the treatment regimen in order to reduce the confounding effect of EGFR-TKIs
miRNAs as therapeutic targets – Future Challenges
In addition to the promising diagnostic, prognostic and predictive value, miRNAs have the potential to become a target themselves for drug development, in order to overcome the acquired resistance to the currently used agents in EGFR-driven lung cancer. Preclinical findings on tumor cell lines clearly demonstrated that both restoring the tumor-suppressor miRNA function (by miRNA mimics) and inhibiting the oncogenic properties of onco-miRs (by antagomiRs) are effective strategies to re-sensitize
Conflicts of interests
The authors have no conflicts of interest, including specific financial interests or relationship and affiliations relevant to the subject matter or materials discussed in the manuscript.
Acknowledgments
This work was supported by Italian Association for Cancer Research (AIRC grant IG2017-20074 to MT, and Start-Up grant to EG).
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