Background: Lung cancer (LC) is still the most common cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all LC cases but is not a single entity. It is now accepted that, apart from the characteristic driver mutations, the unique molecular signatures of adeno-(AC) and squamous cell carcinomas (SCC), the two most common NSCLC subtypes should be taken into consideration for their management. Therapeutic interventions, however, frequently lead to chemotherapy resistance highlighting the need for in-depth analysis of regulatory mechanisms of multidrug resistance to increase therapeutic efficiency. Methods: Non-canonical Wnt5a and canonical Wnt7b and ABC transporter expressions were tested in primary human LC (n = 90) resections of AC and SCC. To investigate drug transporter activity, a three dimensional (3D) human lung aggregate tissue model was set up using differentiated primary human lung cell types. Following modification of the canonical, beta-catenin dependent Wnt pathway or treatment with cisplatin, drug transporter analysis was performed at mRNA, protein and functional level using qRT-PCR, immunohistochemistry, immune-fluorescent staining and transport function analysis. Results: Non-canonical Wnt5a is significantly up-regulated in SCC samples making the microenvironment different from AC, where the beta-catenin dependent Wnt7b is more prominent. In primary cancer tissues ABCB1 and ABCG2 expression levels were different in the two NSCLC subtypes. Non-canonical rhWnt5a induced down-regulation of both ABCB1 and ABCG2 transporters in the primary human lung aggregate tissue model recreating the SCC-like transporter pattern. Inhibition of the beta-catenin or canonical Wnt pathway resulted in similar down-regulation of both ABC transporter expression and function. In contrast, cisplatin, the frequently used adjuvant chemotherapeutic agent, activated beta-catenin dependent signaling that lead to up-regulation of both ABCB1 and ABCG2 transporter expression and activity.

In the aging lung, the lung capacity decreases even in the absence of diseases. The progenitor cells of the distal lung, the alveolar type II cells (ATII), are essential for the repair of the gas-exchange surface. Surfactant protein production and survival of ATII cells are supported by lipofibroblasts that are peroxisome proliferator-activated receptor gamma (PPARγ)-dependent special cell type of the pulmonary tissue. PPARγ levels are directly regulated by Wnt molecules; therefore, changes in the Wnt microenvironment have close control over maintenance of the distal lung. The pulmonary aging process is associated with airspace enlargement, decrease in the distal epithelial cell compartment and infiltration of inflammatory cells. qRT–PCR analysis of purified epithelial and nonepithelial cells revealed that lipofibroblast differentiation marker parathyroid hormone-related protein receptor (PTHrPR) and PPARγ are reduced and that PPARγ reduction is regulated by Wnt4 via a β-catenin-dependent mechanism. Using a human in vitro 3D lung tissue model, a link was established between increased PPARγ and pro-surfactant protein C (pro-SPC) expression in pulmonary epithelial cells. In the senile lung, both Wnt4 and Wnt5a levels increase and both Wnt-s increase myofibroblast-like differentiation. Alteration of the Wnt microenvironment plays a significant role in pulmonary aging. Diminished lipo- and increased myofibroblast-like differentiation are directly regulated by specific Wnt-s, which process also controls surfactant production and pulmonary repair mechanisms.

Background: The predominant metastatic site of lung cancer (LC) is the brain. Although outdated, conventional cisplatin treatment is still the main therapeutic approach for patients with advanced non-small cell lung cancer (NSCLC), since targeted therapy that offers better tumor control is not always possible. In the present study brain metastasis associated cytokine expression was investigated in primary NSCLC adenocarcinoma (AC) tissues with known oncogenic mutations in the presence or absence of platina based and tyrosine kinase inhibitor (TKI) drugs. Methods: Primary lung tumor samples were isolated, DNA was sequenced and then the samples were grouped based on mutation. Experiments were also performed using KRAS mutant A549 and EGFR mutant PC-9 cells. Drug response was analyzed in three dimensional (3D) tissue cultures. We assessed drug response and IL-6 and IL-8 cytokine expression in relation to cellular invasion using ATP dependent cell viability, qRT-PCR analysis, cytokine bead array, and migration assay. Results: In 3D co-cultures, primary NSCLC derived cells harboring EGFR mutation responded better to erlotinib treatment than KRAS mutant or KRAS/EGFR wild type (WT) cancer cells. In contrast, under the same culture conditions KRAS/EGFR WT or KRAS mutant cancer cells are more sensitive to cisplatin than EGFR mutant cells. Drug response and pro-inflammatory cytokine production varied depending on the driver mutations. Cisplatin but not erlotinib increased both IL-6 and IL-8 secretion and only IL-6 increased cellular migration and proliferation. Conclusion: In vitro assays are available to determine the response to planned therapeutic approach of lung cancer subtypes. The sequence of administration of therapeutic drugs determines cytokine production and therefore therapeutic response.

BackgroundAngiogenesis is important both in normal tissue function and disease and represents a key target in lung cancer (LC) therapy. Unfortunately, the two main subtypes of non-small-cell lung cancers (NSCLC) namely, adenocarcinoma (AC) and squamous cell carcinoma (SCC) respond differently to anti-angiogenic e.g. anti-vascular endothelial growth factor (VEGF)-A treatment with life-threatening side effects, often pulmonary hemorrhage in SCC. The mechanisms behind such adverse reactions are still largely unknown, although peroxisome proliferator activator receptor (PPAR) gamma as well as Wnt-s have been named as molecular regulators of the process. As the Wnt microenvironments in NSCLC subtypes are drastically different, we hypothesized that the particularly high levels of non-canonical Wnt5a in SCC might be responsible for alterations in blood vessel growth and result in serious adverse reactions.MethodsPPARgamma, VEGF-A, Wnt5a, miR-27b and miR-200b levels were determined in resected adenocarcinoma and squamous cell carcinoma samples by qRT-PCR and TaqMan microRNA assay. The role of PPARgamma in VEGF-A expression, and the role of Wnts in overall regulation was investigated using PPARgamma knock-out mice, cancer cell lines and fully human, in vitro 3 dimensional (3D), distal lung tissue aggregates. PPARgamma mRNA and protein levels were tested by qRT-PCR and immunohistochemistry, respectively. PPARgamma activity was measured by a PPRE reporter system. The tissue engineered lung tissues expressing basal level and lentivirally delivered VEGF-A were treated with recombinant Wnts, chemical Wnt pathway modifiers, and were subjected to PPARgamma agonist and antagonist treatment.ResultsPPARgamma down-regulation and VEGF-A up-regulation are characteristic to both AC and SCC. Increased VEGF-A levels are under direct control of PPARgamma. PPARgamma levels and activity, however, are under Wnt control. Imbalance of both canonical (in AC) and non-canonical (in SCC) Wnts leads to PPARgamma down-regulation. While canonical Wnts down-regulate PPARgamma directly, non-canonical Wnt5a increases miR27b that is known regulator of PPARgamma.ConclusionDuring carcinogenesis the Wnt microenvironment alters, which can downregulate PPARgamma leading to increased VEGF-A expression. Differences in the Wnt microenvironment in AC and SCC of NSCLC lead to PPARgamma decrease via mechanisms that differentially alter endothelial cell motility and branching which in turn can influence therapeutic response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2943-4) contains supplementary material, which is available to authorized users.

Lung carcinoma is still the most common malignancy worldwide. One of the major subtypes of non-small cell lung cancer (NSCLC) is adenocarcinoma (AC). As driver mutations and hence therapies differ in AC subtypes, we theorized that the expression and function of ABC drug transporters important in multidrug resistance (MDR) would correlate with characteristic driver mutations KRAS or EGFR. Cisplatin resistance (CR) was generated in A549 (KRAS) and PC9 (EGFR) cell lines and gene expression was tested. In three-dimensional (3D) multicellular aggregate cultures, both ABCB1 and ABCG2 transporters, as well as the WNT microenvironment, were investigated. ABCB1 and ABCG2 gene expression levels were different in primary AC samples and correlated with specific driver mutations. The drug transporter expression pattern of parental A549 and PC9, as well as A549-CR and PC9-CR, cell lines differed. Increased mRNA levels of ABCB1 and ABCG2 were detected in A549-CR cells, compared to parental A549, while the trend observed in the case of PC9 cells was different. Dominant alterations were observed in LEF1, RHOU and DACT1 genes of the WNT signalling pathway in a mutation-dependent manner. The study confirmed that, in lung AC-s, KRAS and EGFR driver mutations differentially affect both drug transporter expression and the cisplatin-induced WNT signalling microenvironment.