Development of in vitro 3D culture system to mimic lung cancer tissue

A 3D culture system based on a photocurable matrix has been developed. The aim is to create a 3D printable platform mimicking lung cancer tissue, to study tumor microenvironment evolution, in terms of structural (architecture) and molecular (signalling) components.

Cytoplasmic Localization Isoform of Cyclin Y Enhanced the Metastatic Ability of Lung Cancer via Regulating Tropomyosin 4

Cyclin Y (CCNY) is a novel cyclin and highly conserved in metazoan species. Previous studies from our and other laboratory indicate that CCNY play a crucial role in tumor progression. There are two CCNY isoform which has different subcellular distributions, with cytoplasmic isoform (CCNYc) and membrane distribution isoform (CCNYm). However, the expression and function of CCNY isoforms is still unclear. We firstly found CCNYc was expressed in natural lung cancer tissue and cells through the subcellular distribution. Co-IP and immunofluorescence showed that both CCNYm and CCNYc could interact with PFTK1. Further studies illustrated that CCNYc but not CCNYm enhanced cell migration and invasion activity both in vivo and vitro. The function of CCNYc could be inhibited by suppression of PFTK1 expression. In addition, our data indicated that tropomyosin 4 (TPM4), a kind of actin-binding proteins, was down-regulated by suppression of CCNY. F-actin assembly could be controlled by CCNYc as well as PFTK1 and TPM4. As a result, CCNY was mainly expressed in lung cancer. CCNYc could promote cell motility and invasion. It indicated that CCNYc/PFTK1 complex could promote cell metastasis by regulating the formation of F-actin via TPM4.

Relationship between the miRNA Profiles and Oncogene Mutations in Non-Smoker Lung Cancer. Relevance for Lung Cancer Personalized Screenings and Treatments

Oncogene mutations may be drivers of the carcinogenesis process. MicroRNA (miRNA) alterations may be adaptive or pathogenic and can have consequences only when mutation in the controlled oncogenes occurs. The aim of this research was to analyze the interplay between miRNA expression and oncogene mutation. A total of 2549 miRNAs were analyzed in cancer tissue—in surrounding normal lung tissue collected from 64 non-smoking patients and in blood plasma. Mutations in 92 hotspots of 22 oncogenes were tested in the lung cancer tissue. MicroRNA alterations were related to the mutations occurring in cancer patients. Conversely, the frequency of mutation occurrence was variable and spanned from the k-ras and p53 mutation detected in 30% of patients to 20% of patients in which no mutation was detected. The prediction of survival at a 3-year follow up did not occur for mutation analysis but was, conversely, well evident for miRNA analysis highlighting a pattern of miRNA distinguishing between survivors and death in patients 3 years before this clinical onset. A signature of six lung cancer specific miRNAs occurring both in the lungs and blood was identified. The obtained results provide evidence that the analysis of both miRNA and oncogene mutations was more informative than the oncogene mutation analysis currently performed in clinical practice.

Role of DACH1 on proliferation, invasion, and apoptosis in human lung adenocarcinoma cells

Objective: To investigate DACH1 protein expression in lung cancer tissue and matched paracancerous tissue, and explore its effect on proliferation, invasion, and apoptosis in human lung adenocarcinoma cells (HLACs). Methods: Tumor tissue and matched paracancerous tissue was collected from 46 patients with pathologically diagnosed lung cancer. RT-PCR was perfomed to detect DACH1 mRNA expression and immunohistochemistry to measured DACH1 protein expression. To determine the effect of DACH1 on lung cancer behavior, small interfering RNA (siRNA) was used to silence DACH1 expression in A549 cells. The impact on the proliferation of tumor cells was then observed by MTT assay, changes in the invasion of tumor cells were identified using transwell chamber assay, and the effects on apoptosis in the cell line were detected using flow cytometry. Results: The expression of DACH1 mRNA and DACH1 protein were significantly decreased in lung cancer tissue versus matched paracancerous control tissue. Silencing of DACH1 expression in A549 cells significantly enhanced cell proliferation, significantly increased cell invasion and significantly reduced spontaneous apoptosis. Conclusion: DACH1 is downregulated in lung adenocarcinoma tissue. In vitro assessment shows that DACH1 functions as a tumor suppressor, suggesting its potential use as new target for lung cancer treatment.

Phospholipid dynamics in ex vivo lung cancer and normal lung explants

In cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully 13C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.