Recent Progress 2015

Our group focuses on the identification of the causes, involving non-coding RNAs, behind lung cancer development and resistance to chemotherapy. The most well-known ncRNAs are microRNAs (miRNAs), single stranded RNAs of 19–25 nt in length, that negatively regulate gene expression by translational inhibition or degradation of mRNA targets. MicroRNA dysregulation has been found to be involved in several processes including proliferation, apoptosis and cancer. Our goal is to identify mechanisms of miRNA dysregulation and generate new microRNAs delivery systems to restore normal gene networks in vitro and in vivo improving the efficacy of chemotherapy for lung cancer cure.

MicroRNA biogenesis and function
MicroRNAs or miRNAs are short (20–24-nucleotides) non-coding RNAs, that regulate gene expression at the post-transcriptional level by binding to the 3’-untranslated regions (3’UTRs) or the open reading frames of target genes, leading to the degradation of target mRNA or repression of mRNA translation. MiRNAs are transcribed as long primary transcripts characterised by hairpin structures (pri-miRNAs) whose maturation occurs through sequential processing events (Gregory 2004) (Figure. 1). The mature miRNAs are incorporated into a complex named RISC (RNA-induced silencing complex), which contains Argonaute proteins. The function of the miR is to guide the RISC to complementary or partially complementary target sites located in the 3’ UTRs of mRNAs target inducing mRNA degradation or block of translation, respectively. The mechanism of action of microRNAs has revolutionised the concept of gene expression regulation, because we now know that mRNA levels in a cell do not strictly correlate with protein expression (Bartel 2004). miRNAs are predicted to regulate a total of ~60% of human genes. A single miRNA can act on different mRNA targets whereas multiple miRNAs can regulate single mRNA molecules (Bartel 2004). As more miRNAs are unravelled and their role identified, it becomes clear that the involvement of these molecules in cancer is much more extensive than initially thought (Kasinsky and Slack 2001). The most striking evidence of the involvement of microRNAs in cancer is the alteration of miRNA expression in malignant cells compared to the normal counterparts (Calin and Croce 2006). The specific miRNA signature, called miRNome, characterises the malignant state and defines the clinicopathological features of the tumours (e.g. stage, grade, aggressiveness, proliferation index). Several high-throughput technologies revealed that miRNA stratification can be easily used to classify tumours and predict patient outcome. Because a single miRNA can target multiple pathways, miRNA-based anticancer therapies are being developed, either alone or combined with chemotherapy to improve the response and increase cure rates.

Figure 1. miRNA biogenesis and function. The primary miRNA (pri-miRNA) is transcribed by the RNA pol II from its genomic location and cleaved by the microprocessor complex, which comprises Drosha and DGCR8. The resulting pre-miRNA is transported by the exportin 5 to the cytoplasm where it is further processed into a double strand mature miRNA by Dicer and its cofactors. One strand of this duplex is degraded, whereas the other strand accumulates as the mature microRNA, which binds and guides the protein effector complex, formed by the RNA-induced silencing complex (RISC) and miRgonaute, to messenger RNA targets inducing either block of translation or mRNA degradation. 

MicroRNAs and chemoresistance
Lung cancer still represents a very deadly disease in strong need of new, effective, therapeutic approaches. The long-term survival for patients with advanced high-grade lung cancer has been limited by the frequent occurrence of resistance to chemotherapeutic drugs. In this context, TRAIL may represent an alternative therapeutic molecule for this type of cancer. Several TRAIL inhibitors have entered clinical trials and seem to be effective in a small fraction of lung cancer patients (Lorusso et al., 2012; Herbst, 2010). However, as with other molecularly targeted agents, resistance is likely to develop. Acquired apoptosis resistance is detrimental not only because it dampens the anticancer activity of the drugs but also because it promotes cancer progression and metastasis (Malhi 2006). The molecular mechanisms underlying the resistant TRAIL phenotype is still unclear and little is known regarding how lung cancer can acquire resistance to TRAIL. Therefore, it is fundamental to identify biomarkers to predict the response to the drug and to improve its therapeutic efficacy using drug combinations that not only synergise with TRAIL but that might also overcome resistance as it arises. To this end, we generated TRAIL-resistant cells (H460R and H292R) by exposing H460 and H292 sensitive cells (H460S and H292S) to stepwise increases in TRAIL concentrations over a period of six months to select cells capable of growing at high concentrations of TRAIL. miRNA expression profile in H460R versus H460S revealed dysregulation of a set of microRNAs (Jeon et al., under review). We found that these microRNAs are transcriptionally regulated by NF-kB and modulate important tumour suppressor genes involved in the TRAIL pathway. A combination of TRAIL and NF-KB inhibitors has shown, in vitro and in vivo, increased apoptosis and reduced cell proliferation in TRAIL resistant cells compared to treatments involving TRAIL or NF-KB inhibitors alone. The results not only suggest that combinatory treatment of TRAIL and NF-KB inhibitors could be effective in overcoming TRAIL resistance in NSCLC but also that, in the near future, the delivery and modulation of specific microRNAs could improve the response of  lung cancer patients to TRAIL.

Our future plans involve the study of TKR-regulated-microRNAs. TKRs have shown a crucial role in several tumours, including lung cancer (Garofalo 2012). Currently, the epidermal growth factor receptor (EGFR) inhibitors represent the standard of care for patients with locally advanced or metastatic NSCLC harbouring activating EGFR mutations. Other genetic abnormalities have been reported in several small distinct subsets of NSCLC. Among these rare genetic changes, anaplastic lymphoma kinase (ALK) gene rearrangements result in the abnormal expression and activation of this tyrosine kinase. These rearrangements occur in 2-5% of NSCLC. Crizotinib, a first-in-class dual ALK and c-MET inhibitor, has been shown to be particularly effective against ALK positive NSCLC, showing dramatic and prolonged responses with low toxicity. However, resistance to crizotinib inevitably emerges and the mechanisms of such resistance are unknown. In this context we plan to investigate the role of non-coding RNAs in crizotinib resistance in vitro and in vivo. Plasma microRNAs will be assessed using microfluidic array technology in a screening cohort of healthy controls and crizotinib sensitive and resistant patients as noninvasive biomarkers to predict the response to the drug.

MicroRNAs and the microenvironment
The communication between the tumour cells and the surrounding cells—the microenvironment—helps drive the process of tumour progression. Two of the key hallmarks of cancer, angiogenesis and metastasis, are dependent on the surrounding microenvironment. Exosomes function as mediators of intercellular communication and contain multiple functional molecules including microRNAs. We plan to study how exosome-released microRNAs influence the communication between normal and malignant cells to induce lung tumorigenesis. Networks of pro-metastatic regulators involved in angiogenesis, collagen remodelling and proteolysis will be analysed as targets of the secreted microRNAs. Understanding molecular signalling in the tumour microenvironment may provide new mechanistic rationales for optimising current cancer therapies and the development of future novel therapeutic modalities.