Current status of NSCLC

Lung cancer is a serious public health problem that affects the lives of millions and is the most common cause of cancer-related mortality [1]. Moreover, according to WHO projections, the global burden of lung cancer is expected to rise due to industrial and environmental pollution and many other factors. In 2010, a report from National Central Cancer Registry of China demonstrated that the lung cancer incidence and mortality increased to 46.08/100,000 and 37.00/100,000, respectively [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. NCI SEER database reported that 5-year survival was 58.2% for NSCLC patients diagnosed at localized stage (18%) of all stages including localized, regional and distant from 2004–2010, whereas it was only 4.5% when diagnosed at distant stage (55%) [3]. Although many advances achieved in cancer biology as well as in diagnosis and treatment, progress in lung cancer therapy has been slow, leading to about 5% improvement in 5-year survival rates for the last 20 years [4].

Most important advances include the discovery of oncogenic driver genes and therapies specific for these genes or pathways. The common driver genes identified so far in NSCLC included the mutant activations of KRAS, EGFR, Met and BRAF [5]. In comparison with standard chemotherapy, new target therapies show a significant improved progression-free survival (PFS) and relatively less toxicity [4]. However, the median PFS of NSCLC patients with EGFR mutation was only 9.5 months on treatment with tyrosine kinase inhibitors (TKIs) [6]; The median PFS for patients with locally advanced ALK-positive lung cancer was only 7.7 months with crizotinib treatment [7]. Moreover, the overall survivals are not significantly improved for patients received target therapy as compared with chemotherapy [6]-[8]. It is obviously that overcoming refractory to target drugs can prolong treatment response. In this respect, experimental study proved that inhibition of hedhehog signaling abrogated resistance of NSCLC to TKI [9].

Novel inhibitors targeting multiple kinase sites are extensively developed, some have entered clinical trial. For example, a novel kinase inhibitor targeting both EGFR and HER2 has been evaluated in lung and breast cancer in phase I study and is expected to bring improved response [10]. Meanwhile, combination of target drugs with traditional chemotherapeutics had also been deployed, such as the conjunction of MEK inhibitors with anti-cancer drug docetaxel in previously treated patients with advanced lung cancer. Combined therapy revealed better response rate and prolonged PFS than chemotherapy alone in a phase II randomized trial [11].

In translational research, scientists performed comprehensive molecular studies to search for new genes in lung cancer. Whole-genome and transcriptome sequencing of tumor and adjacent normal tissue samples from NSCLC had identified novel alterations in genes involved in chromatin modification and DNA repair pathways, novel metabolic enzymes, as well as aberration of cell fate determination factor DACH1 [96]. Gifitinib-acquired resistant lung cancer cells displayed EMT phenotype. Molecular analyses confirmed that activation of Notch-1 and its target genes, which induced expression of mesenchymal marker vimentin, snail and hes-1, and decreased epithelial marker E-cadherin. The elevated expression of Notch-1 could directly suppress p21 Waf1/Cipl accompanied with the increasing of cyclin D1, giving rise to the EMT phenotype. Meanwhile, knockdown of Notch-1 decreased cyclinD1 expression and reversed EMT. These findings suggest that the aggressive behavior caused by cyclinD1 might be driven by the mis-expression of Notch pathway in NSCLC, which finally down regulates p21 Waf1/Cipl and promotes the EMT phenotype [71].

Targeting Notch signaling in NSCLC

In vivo evidence that Notch inhibitor is a potential therapeutic agent came from Kras(G12V)-driven NSCLCs mice model. Pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocked cancer growth. Correspondingly, molecular analysis of treated cancer tissues demonstrated reduced HES1 levels and phosphorylated ERK [97]. Currently, several classes of investigational Notch inhibitors have been developed. These include monoclonal antibodies against Notch receptors or ligands, decoys to soluble forms of the extracellular domain of Notch receptor or Notch ligands, blocking peptides, and gamma-secretase inhibitors (GSIs) or natural compounds [98].

At present, GSIs are the most extensively explored. RO4929097, a small-molecule inhibitor of GSI with high oral bioavailability and is a potent and selective inhibitor of gamma-secretase, has been tested in phase I study in refractory metastatic or locally advanced solid tumors [99], and phase II studies for metastatic melanoma [100], metastatic colorectal cancer [101] and metastatic pancreatic adenocarcinoma [102]. Another GSIs, PF-03084014, was also evaluated on phase I in advanced solid tumor [103]. In preclinical study, MRK-003 was evaluated in triple negative breast cancer cells by MRK-003 alone and in combination with paclitaxel. Immunohistochemical staining for activated NOTCH1 and HES4 expression could be molecular biomarkers, identifying solid tumors that are likely to respond to GSI-based therapies [104]. Preclinical study of MRK-003 in pancreatic cancer [105] and in multiple myeloma and non-Hodgkin’s lymphoma exhibited promising activity [106]. Treatment with GSIs MK-0752 in breast cancer cell lines reduced stem cell subpopulation in vitro and in human tissues from clinical trial [107]. Clinical benefit of MK-0752 in adult patients with advanced solid tumors was observed with well tolerated toxicity in Phase I study, therefore promoting to combinational trials [108].

Inhibitors of Notch signaling can be used not only as direct anti-cancer agents but also as a sensitizer to current therapy. Platinum-based chemotherapy is the first-line treatment for NSCLC, but recurrence occurs in most patients. Experimental study found that treatment of NSCLC cell line H460 and H661 enriched CD133 (+) cells and upregulated ABCG2 and ABCB1 expression, which conferred the cross-resistance to doxorubicin and paclitaxel. Detailed molecular analysis found that the enrichment of CD133 (+) cells by cisplatin depended on Notch signaling. Moreover, pretreatment with the γ-secretase inhibitor or Notch1 short hairpin RNAs (shRNA) remarkably increased the sensitivity to doxorubicin and paclitaxel. Importantly, similar phenomena were observed both in engrafted tumors derived from transplanted animal model and the relapsed tumors of patients who had received cisplatin treatment [109]. Gamma-secretase inhibitor DAPT alone slightly inhibited the proliferation and exhibited little effect on the cell cycle, but enhanced the inhibitory effects of Cisplatin in a combinational study with GSI. Interestingly, this effect was especially significant in CD133 (+) cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer [110]. In complementary, Dr. Carbone’s group found that treatment of EGFR-mutated lung cancer cell lines with erlotinib enriched the ALDH+ stem-like cells with stem-like cell potential through EGFR-dependent activation of Notch3. Moreover, γ secretase inhibitor could reverse this phenotype. At molecular level, physical association between the Notch3 and EGFR receptors leads to tyrosine phosphorylation of Notch3. This study could explain the unflavored survival observed in some studies of erlotinib treatment at early-stage disease, and imply that specific dual targeting might overcome adverse effect of TKIs [111].

γ-Secretase inhibitor administration after radiation had the greatest growth inhibition of lung cancer in vitro and in vivo. Mechanically, enhanced apoptosis of lung cancer cell lines in combination group were through regulation of MAPK and Bcl-2 family proteins. Furthermore, radiation-induced activation of Notch was blocked by GSI administration, suggesting that treatment with GSI could prevent Notch-induced radiation resistance [112]. Together, above studies provided compelling evidence for exploiting Notch inhibitors in clinic trial. In most cases, the application of GSI is limited because of gut toxicity and goblet cell hyperplasia. Therefore, the best dose and schedule need to be optimized.

Other receptor-specific approaches like siRNA directed against Notch might be useful in reducing the tumorigenicity and invasive of NSCLC. For instance, nanoparticle (NP) technology has been applied to deliver specific siRNA to knockdown Notch1 to arrest tumor growth and reverse EMT by the up-regulation of miR-200a and down-regulation of the transcription factors ZEB1, ZEB2, Snail and Slug [113]. Furthermore, monoclonal antibody against Notch can also reduce the invasion of NSCLC [114],[115]. The complex of γ-secretase is a transmembrane protease that catalyses the cleavage of a set of membrane proteins and is comprised of four subunits encoded by four genes, including PSEN1, PSENEN, NCSTN and APH1. Targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. It is suggested that measuring NCSTN in patient samples may serve as a molecular marker for anti-NCSTN therapy in the clinic [116].

>Some natural agents such as curcumin, 3,3′-diindolylmethane (DIM), resveratrol, and epigallocatechin-3-gallate (EGCG) have been reported to be effective on targeting Notch signaling, suggesting they can also be promising alternative strategies for NSCLC chemotherapy [117],[118].

Conclusion

As a well known driving force of cell invasion, migration and metastasis, EMT has been shown to induce resistance of cancer cells to conventional chemotherapy and radiotherapy. Notch signaling not only activates cell proliferation, antagonize apoptosis but also cross-talks with several transcriptional factors to promote EMT, leading to enhanced motility in vitro and invasion and metastasis in vivo. Theoretically, combination of chemotherapy or radiotherapy with Notch inhibitors might acquire synergistic effect and improve chemotherapy response. Although promising results have been noticed in some patients with Notch inhibitors in clinical trials, stratification biomarkers to identify patients who are most likely benefit from GSIs treatment are required for a successful development of this class of drugs.