Abstract
Macrophage polarization and infiltration to the tumor microenvironment (TME) is a critical determining factor for tumor progression. Macrophages are polarized into two states—M1 (pro-inflammatory, anti-tumorigenic and stimulated by LPS or IFN-γ) and M2 (anti-inflammatory pro-tumorigenic and stimulated by IL-4) phenotypes. Specifically, M2 macrophages enhance tumor cell growth and survival. Recent evidences suggest the pivotal role of microRNAs in macrophage polarization during the development of Non-small cell lung cancer (NSCLC), thus proposing a new therapeutic option to target lung cancer. In silico analysis determined cogent upregulation of KLF4, downregulation of IL-1β and miR-34a-5p in NSCLC tissues, consequently worsening the overall survival of NSCLC patients. We observed a significant association of KLF4 with macrophage infiltration and polarization in NSCLC. We found that KLF4 is critically implicated in M2 polarization of macrophages, which, in turn, promotes tumorigenesis. KLF4 expression correlated with miR-34a-5p and IL-1β in a feed-forward loop (FFL), both of which are implicated in immune regulation. Mechanistic overexpression of miR-34a-5p in macrophages (IL-4 stimulated) inhibits KLF4, along with downregulation of ARG1, REL-1MB (M2 macrophage specific markers), and upregulation of IL-1β, IL-6, (M1 macrophage specific markers), demonstrating macrophage polarization switch from M2 to M1 phenotype. Moreover, co-culture of these macrophages with NSCLC cells reduces their proliferation, wound healing, clonogenic capacity and enhanced NO-mediated apoptosis. Further, transfection of miR-34a-5p in NSCLC cells, also degrades KLF4, but enhances the expression of KLF4 regulated genes—IL-1β, IL-6 (pro-inflammatory mediators), which is further enhanced upon co-culture with IL-4 stimulated macrophages. Additionally, we observed a significant increase in i-NOS/NO content upon co-culture, suggesting polarization reversion of macrophages from M2 to M1, and eventually leading to anti-tumor effects. Our findings thus show a significant role of KLF4 in tumorigenesis and TAM polarization of NSCLC. However, miR-34a-5p mediated targeting of these molecular networks will provide a better therapeutic intervention for NSCLC.
Highlights
Lung cancer (LC) is one of the prime causes of cancer-associated mortality worldwide
The list of Transcription factors (TFs) were scrutinized via literature studies and only Non-small cell lung cancer (NSCLC)-specific TFs were retained miRNA-gene/TF repression: miRNA-gene/TF pairs were extracted from miRWalk v3.0 [41], miRSearch v3.0 [42] and Starbase v2.0 [43] databases, respectively
In consideration with the abovementioned threshold, a total of 612 differentially expressed genes (DEGs) and 467 differentially expressed miRNAs (DEMs) were identified from datasets GSE75037 and GSE53882, respectively
Summary
Lung cancer (LC) is one of the prime causes of cancer-associated mortality worldwide. The expression profiles of cytokines and chemokines determine the depth of immune responses and inflammatory reactions [6,7,8,9,10] Both the phenotypes represent the extremities of TAM function, with M2 phenotype, being immensely associated with initiation, development, progression, and poor prognosis of cancers [11,12,13]. KLF4 is a highly conserved nuclear TF, containing a zinc-finger like domain, bearing N-terminal transcriptional activation domain, C-terminal transcription inhibitory region along with nuclear localization sequences for the regulation of protein interactions [19] It is critically intricated into several aspects of cellular processes, including proliferation, differentiation, somatic reprogramming, tissue homeostasis and apoptosis [20]. Reversal of TAM polarization promotes antitumor activities in NSCLC
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have