Smart delivery of lethal poly-peptide composite for effective cancer therapy

In the past decade, multifunctional peptides have attracted increasing attention in the biomedical field. Peptides possess many impressive advantages, such as high penetration ability, low cost, and etc. However, the short half-life and instability of peptides limit their application. In this study, a poly-peptide drug loading system (called HKMA composite) was designed based on the different functionalities of four peptides. The peptide compositions of HKMA composite from N-terminal to C-terminal were HCBP1, KLA, MMP-2-cleavable peptide and ABD. The targeting and lethality of HKMA to NSCLC cell line H460 sphere cells and the half-life of the system were measured in vivo. The results showed that the HKMA composite had a long half-life and specific killing effect on H460 sphere cells in vitro and in vivo. Our result proposed smart peptide drug loading system and provided a potential methodology for effective cancer treatment.

CRABP2 involvement in a mechanism of Golgi stress and tumor dry matter in non-small cell lung cancer cells via ER dependent Hippo pathway

Objective: The paper aimed to explore the mechanism of cellular retinoic acid binding protein 2 (CRABP2) involvement in Golgi stress and tumor dryness in non-small cell lung cancer (NSCLC) cells through the estrogen receptor (ER) dependent Hippo pathway. Methods: Human NSCLC cell line A549 was purchased from ATCC andcultured in RPMI-1640 with 10% FBS. Attractene reagent was used for plasmid transfection. ER (sh) RNA was designed using RNAi Designer. Seventy-six hours after infection, stable cells were obtained after treated with puromycin for 3 weeks. ER silencing cells (with inhibited ER expression) were compared to the control cells (normal cultured NSCLC cell line A549, CRABP2 normal expression). CRABP2 and ER expression levels were detected by RT-PCR. MTT assay was used to detect cell proliferation, and the cell localization of ER and Golgi was observed by confocal microscopy. The invasion and metastasis of cells were analyzed by Boden chamber invasion and migration assays. Western blotting assays was used for detecting the protein expression of E-cadherin, vimentin, ZO-1 protein and epithelial-mesenchymal transition (EMT) related factors. Results: The lower expression level of mRNA was detected in the ER-silencing group compared to the control group (P<0.05). We also found a higher proliferation level of cells, the number of invading and metastatic cells, the expression of vimentin, p-Lats1T1079, Lats1 and p-YAPS127 mRNA in the control group compared to the ER silencing group (P<0.05). And the expression level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2 alpha), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) in the control group was higher than that in the ER silencing group (P<0.05). Adversely, a lower expression level of E-cadherin and ZO-1 protein was found in the control group compared to the ER silencing group (P<0.05). Conclusion: The expression of CRABP2 in NSCLC cells was regulated by ER, and cell proliferation and invasion were regulated by the Hippo pathway. At the same time, it was found that decreased expression of CRABP2 enhanced endoplasmic reticulum/Golgi stress response.

PD-L1 blockade exhibits anti-tumor effect on brain metastasis by activating CD8+ T cells in hematogenous metastasis model with lymphocyte infusion

Brain metastases are common complication in cancer patients. Immune checkpoint inhibitors show therapeutic benefits also in patients with central nervous system (CNS) metastases. However, their antitumor effects on metastatic tumors and their underlying mechanisms are still poorly understood. In this study we investigated the antitumor effect of anti-programmed death-ligand 1 (PD-L1) antibody on metastatic brain tumors and evaluated immune responses during treatment. We employed a hematogenous brain metastasis xenograft model using immunodeficient mice with murine lymphocyte infusions. A human non-small-cell lung cancer (NSCLC) cell line stably expressing NanoLuc® reporter (Nluc-H1915) was inoculated from the internal carotid artery of SCID mice. After metastases were established (24 days after inoculation), splenocytes prepared from H1915-immunized BALB/c mice were injected intravenously and mouse IgG or anti-PD-L1 antibody treatment was started (day 1). Evaluated by Nluc activity, tumor volume in the brain on day 14 was significantly lower in anti-PD-L1-treated mice than in mouse IgG-treated mice. Furthermore CD8+ cells were primarily infiltrated intratumorally and peritumorally and anti-PD-L1 treatment induced a significantly higher proportion of Granzyme B (GzmB)+ cells among CD8+ T cells. The antitumor effect of anti-PD-L1 antibody on brain metastasis is thought to be achieved by the enhanced activation of infiltrated CD8+ T cells into metastatic brain tumor. These results suggest that anti-PD-L1 antibody-containing regimens may be promising treatment options for cancer patients with brain metastases.

GCC2 as a New Early Diagnostic Biomarker for Non-Small Cell Lung Cancer

No specific markers have been identified to detect non-small cell lung cancer (NSCLC) cell-derived exosomes circulating in the blood. Here, we report a new biomarker that distinguishes between cancer and non-cancer cell-derived exosomes. Exosomes isolated from patient plasmas at various pathological stages of NSCLC, NSCLC cell lines, and human pulmonary alveolar epithelial cells isolated using size exclusion chromatography were characterized. The GRIP and coiled-coil domain-containing 2 (GCC2) protein, involved in endosome-to-Golgi transport, was identified by proteomics analysis of NSCLC cell line-derived exosomes. GCC2 protein levels in the exosomes derived from early-stage NSCLC patients were higher than those from healthy controls. Receiver operating characteristic curve analysis revealed the diagnostic sensitivity and specificity of exosomal GCC2 to be 90% and 75%, respectively. A high area under the curve, 0.844, confirmed that GCC2 levels could effectively distinguish between the exosomes. These results demonstrate GCC2 as a promising early diagnostic biomarker for NSCLC.

Contribution of Neuropilin-1 in Radiation-Survived Subclones of NSCLC Cell Line H1299

Non-small cell lung cancer (NSCLC) is an aggressive lung cancer accounting for approximately 85% of all lung cancer patients. For the patients with Stages IIIA, IIIB, and IIIC, the 5-year survival is low though with the combination with radiotherapy and chemotherapy. In addition, the occurrence of tumor cells (repopulated tumors) that survive irradiation remains a challenge. In our previous report, we subcloned the radiation-surviving tumor cells (IR cells) using the human NSCLC cell line, H1299, and found that the expression of neuropilin-1 (NRP-1) was upregulated in IR cells by the microarray analysis. Here, we investigated the contribution of neuropilin-1 to changes in the characteristics of IR cells. Although there were no differences in angiogenic activity in the tube formation assay between parental and IR cells, the cell motility was increased in IR cells compared to parental cells in the cell migration assay. This enhanced cell motility was suppressed by pretreatment with anti-NRP-1 antibody. Although further studies are necessary to identify other molecules associated with NRP-1, the increase in cellular motility in IR cells might be due to the contribution of NRP-1. Inhibition of NRP-1 would help control tumor malignancy in radiation-surviving NSCLC.

SYST-02. DUAL INHIBITION OF BCL-2 AND EGFR IN A PRECLINICAL MODEL OF LUNG CANCER BRAIN METASTASIS

Lung cancer is the most prevalent malignancy to affect both men and women. Around 80% of all lung cancers are classified as non-small cell lung cancer (NSCLC). This subtype of lung cancer is also the most likely to metastasize to the brain. Clinically, the common treatment for NSCLC is epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), due to the high occurrence of EGFR mutations. However, the cancer cells quickly develop resistance to the EGFR TKIs. This resistance and the added difficulty of delivering drugs across the blood-tumor barrier in efficacious concentrations to treat brain lesions are important to consider when developing treatment strategies for lung cancer brain metastases. Our study utilizes a NSCLC cell line, PC-9-Br6, which was developed in our laboratory to preferentially metastasize to the brain. This cell line was demonstrated by our collaborator to express higher levels of Bcl-2 in comparison to the parental PC-9-P cell line. We hypothesized combining novel Bcl-2 inhibitors (ABT-199/ABT-263) with an EGFR inhibitor (gefitinib) would increase survival and decrease tumor burden in our clinically relevant mouse model of lung cancer brain metastases.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. In recent work we identified mutations in Protein Tyrosine Phosphatase Receptor Type H (Ptprh, also known as SAP-1) as being associated with elevated phosphorylation of EGFR in a mouse model of breast cancer. We have examined a series of tumors from this mouse model, revealing conserved V483M Ptprh mutations within the FVB background, but a series of varied mutations in other backgrounds. Despite the varied Ptprh mutations in other background strains, matched primary and metastatic tumors largely shared mutational profiles. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while a rescue with a D986A catalytically dead mutant PTPRH did not, demonstrating that PTPRH targets EGFR. As expected with active EGFR, the knockout of PTPRH was associated with increased growth rate. Moreover, a dose response curve illustrated that two human NSCLC lines that had naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Injection of one of the NSCLC human lines into mice resulted in tumors, and Osimertinib treatment resulted in a reduction of tumor volume relative to vehicle controls. Consistent with prior literature from breast cancer, PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Other roles for PTPRH were explored through a receptor tyrosine kinase array, noting elevated phosphorylation of FGFR1. Knockout of PTPRH in NSCLC cell lines resulted in elevated phosphorylated FGFR1 relative to controls, indicating that PTPRH has a number of targets that may be aberrantly activated in NSCLC with mutations in PTPRH. Together these data suggest that mutations in PTPRH in NSCLC may result in clinically actionable alterations using existing therapies.

High-Throughput Screen of Natural Compounds and Biomarkers for NSCLC Treatment by Differential Expression and Weighted Gene Coexpression Network Analysis (WGCNA)

Lung cancer is known as the leading cause which presents the highest fatality rate worldwide; non-small-cell lung cancer (NSCLC) is the most prevalent type of lung carcinoma with high severity and affects 80% of patients with lung malignancies. Up to now, the general treatment for NSCLC includes surgery, chemotherapy, and radiotherapy; however, some therapeutic drugs and approaches could cause side effects and weaken the immune system. The combination of conventional therapies and traditional Chinese medicine (TCM) significantly improves treatment efficacy in lung cancer. Therefore, it is necessary to investigate the chemical composition and underlying antitumor mechanisms of TCM, so as to get a better understanding of the potential natural ingredient for lung cancer treatment. In this study, we selected 78 TCM to treat NSCLC cell line (A549) and obtained 92 transcriptome data; differential expression and WGCNA were applied to screen the potential natural ingredient and target genes. The sample which was treated with A. pierreana generated the most significant DEG set, including 6130 DEGs, 2479 upregulated, and 3651 downregulated. KEGG pathway analyses found that four pathways (MAPK, NF-kappa B, p53, and TGF-beta signaling pathway) were significantly enriched; 16 genes were significantly regulated in these four pathways. Interestingly, some of them such as EGFR, DUSP4, IL1R1, IL1B, MDM2, CDKNIA, and IDs have been used as the target biomarkers for cancer diagnosis and therapy. In addition, classified samples into 14 groups based on their pharmaceutical effects, WGCNA was used to identify 27 modules. Among them, green and darkgrey were the most relevant modules. Eight genes in the green module and four in darkgrey were identified as hub genes. In conclusion, we screened out three new TCM (B. fruticose, A. pierreana, and S. scandens) that have the potential to develop natural anticancer drugs and obtained the therapeutic targets for NSCLC therapy. Our study provides unique insights to screen the natural components for NSCLC therapy using high-throughput transcriptome analysis.

Gene expression profiling after LINC00472 overexpression in an NSCLC cell line

Lung cancer accounts for a large proportion of cancer-related deaths worldwide. Personalized therapeutic medicine based on the genetic characteristics of non-small cell lung cancer (NSCLC) is a promising field, and discovering clinically applicable biomarkers of NSCLC is required. LINC00472 is a long non-coding RNA and has been recently suggested to be a biomarker of NSCLC, but little is known of its mechanism in NSCLC. Thus, the current study was performed to document changes in gene expression after LINC00472 overexpression in NSCLC cells. As a result of cell viability and migration assay, LINC00472 downregulated cell survival, proliferation, and motility. Transcriptome sequencing analysis showed 3,782 genes expression were changed in LINC00472 overexpressing cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed most genes were associated with intracellular metabolism. The PPP1R12B, RGS5, RBM5, RBL2, LDLR and PTPRM genes were upregulated by LINC00472 overexpression and these genes functioned as tumor suppressors in several cancers. In contrast, SPSB1, PCNA, CD24, CDK5, CDC25A, and EIF4EBP1 were downregulated by LINC00472, and they functioned as oncogenes in various cancers. Consequently, the function of LINC00472 in tumorigenesis might be related to changes in the expressions of other oncogenes and tumor suppressors.

Tin Mesoporphyrin Selectively Reduces Non-Small-Cell Lung Cancer Cell Line A549 Proliferation by Interfering with Heme Oxygenase and Glutathione Systems

In order to maintain redox homeostasis, non-small-cell lung cancer (NSCLC) increases the activation of many antioxidant systems, including the heme-oxygenase (HO) system. The overexpression of HO-1 has been often associated with chemoresistance and tumor aggressiveness. Our results clearly showed an overexpression of the HO-1 protein in A549 NSCLC cell lines compared to that in non-cancerous cells. Thus, we hypothesized that “off-label” use of tin mesoporphyrin, a well-known HO activity inhibitor clinically used for neonatal hyperbilirubinemia, has potential use as an anti-cancer agent. The pharmacological inhibition of HO activity caused a reduction in cell proliferation and migration of A549. SnMP treatment caused an increase in oxidative stress, as demonstrated by the upregulation of reactive oxygen species (ROS) and the depletion of glutathione (GSH) content. To support these data, Western blot analysis was performed to analyze glucose-6-phosphate dehydrogenase (G6PD), TP53-induced glycolysis and the apoptosis regulator (TIGAR), and the glutamate cysteine ligase catalytic (GCLC) subunit, as they represent the main regulators of the pentose phosphate pathway (PPP) and glutathione synthesis, respectively. NCI-H292, a subtype of the NSCLC cell line, did not respond to SnMP treatment, possibly due to low basal levels of HO-1, suggesting a cellular-dependent antitumorigenic effect. Altogether, our results suggest HO activity inhibition may represent a potential target for selective chemotherapy in lung cancer subtypes.