Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non-small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD-1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti-CTLA4 antibody can induce PD-L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti-CTLA4 antibody was not able to induce PD-L1 expression in NSCLC cells. Moreover, anti-CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell-intrinsic CTLA4 can regulate PD-L1 expression and cell proliferation, and that anti-CTLA4 antibody, by binding to the tumour cell-intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.

Background

Aberrant JAK/STAT activation has been detected in many types of human cancers. The role of JAK/STAT activation in cancer has been mostly attributed to direct transcriptional regulation of target genes by phosphorylated STAT (pSTAT), while the unphosphorylated STAT (uSTAT) is believed to be dormant and reside in the cytoplasm. However, several studies have shown that uSTATs can be found in the nucleus. In addition, it has been shown that tissue-specific loss of STAT3 or STAT5 in mice promotes cancer growth in certain tissues, and thus these STAT proteins can act as tumor suppressors. However, no unifying mechanism has been shown for the tumor suppressor function of STATs to date. We have previously demonstrated a non-canonical mode of JAK/STAT signaling for Drosophila STAT and human STAT5A, where a fraction of uSTAT is in the nucleus and associated with Heterochromatin Protein 1 (HP1); STAT activation (by phosphorylation) causes its dispersal, leading to HP1 delocalization and heterochromatin loss.

Methods

We used a combination of imaging, cell biological assays, and mouse xenografts to investigate the role of STAT3 in lung cancer development.

Results

We found that uSTAT3 has a function in promoting heterochromatin formation in lung cancer cells, suppressing cell proliferation in vitro, and suppressing tumor growth in mouse xenografts.

Conclusions

Thus, uSTAT3 possesses noncanonical function in promoting heterochromatin formation, and the tumor suppressor function of STAT3 is likely attributable to the heterochromatin-promoting activity of uSTAT3 in the non-canonical JAK/STAT pathway.

Being a link between a uniaxial nematic and a chiral nematic, the twist bend nematic (NTB) has been an intriguing topic for research over the last decade as a key to understanding chirality generation. Accurate description and understanding of resonance effects in helical structures provide crucial knowledge on the phase behavior beyond positional ordering. We examined the manifestation of resonance effects in NTBviatender resonant X-ray scattering (TReXS) at the sulfur K-edge. For the first time we demonstrate quantitatively that the energy dependence of the scattering peak in the NTBphase follows the energy dependence of the complex refractive indices measured by X-ray absorption. Taking advantage of molecular energy calculation, we decipher the distinct helical pitch variation trend for symmetric/asymmetric dimers from the perspective of hybridization and reveal the strong effect of substituting oxygen for sulfur atoms on the helical pitch and the thermal helix extension.

Human immunodeficiency virus type 1 (HIV-1) is responsible for the majority of HIV infections worldwide, and we still lack a cure for this infection. Blocking the interaction of HIV-1 and its primary receptor CD4 is one strategy for identifying new anti-HIV-1 entry inhibitors. Here we report the discovery of a novel ligand that can inhibit HIV-1 entry and infection via CD4. Biological and computational analyses of this inhibitor and its analogs, using bioactivity evaluation, Rule of Five (RO5), comparative molecular field analysis (CoMFA)/comparative molecular similarity index analysis (CoMSIA) models, and three-dimensional quantitative structure-activity relationship (3D-QSAR), singled out compound 3 as a promising lead molecule for the further development of therapeutics targeting HIV-1 entry. Our study demonstrates an effective approach for employing structure-based, rational drug design techniques to identify novel antiviral compounds with interesting biological activities.

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model of GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and binding free energy calculation to determine how the peptide interacts with CXCR4 and inhibits its downstream signaling. These results demonstrate that this combinational approach is effective for generating nanomolar active inhibitors of CXCR4 and may be applicable to other GPCRs.