BACKGROUND:Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases.MATERIALS AND METHODS:Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259.RESULTS:Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases.CONCLUSIONS:This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

mesothelioma (MPM).We investigated the associations between physical function, symptom burden, and QoL in order to identify key factors that may be targeted for supportive care interventions.Methods: This was a prospective, cohort study of MPM patients enrolled consecutively at a single center.Baseline metrics were obtained at the first visit and repeated every 3 months for up to 5 visits.Symptom and QoL metrics were assessed via the EORTC QLQ-C30 questionnaire.Lower extremity function was measured with the Short Physical Performance Battery (SPPB), which includes balance, gait, and chair stand tests.Spearman rank correlation was used to examine baseline relationships between physical function and symptom and QoL measures.Univariate and multivariate Cox proportional hazards models were used to calculate hazard ratios for all-cause mortality.Results: Seventy-six patients were enrolled between May 2019 and March 2020.Median age was 70 (range 41-89); 70% were male; 92% were White (non-Hispanic); 76.3% had epithelioid histology.The prevalence of baseline symptoms were: fatigue (80.3%), dyspnea (72.4%), insomnia (63.2%), pain (58.7%), constipation (39.5%), appetite loss (36.8%), nausea (23.7%), and diarrhea (17.1%).The mean EORTC global health status/QoL score was 67.2 (SD 19.7), mean EORTC summary score was 78.3 (SD 15.4), and mean SPPB score was 10.7 (SD 1.5).Total SPPB score was significantly correlated with global health status/QoL, summary health, fatigue, dyspnea, and appetite loss (rj.35j, p<.01), but not with pain or insomnia.These correlations were preserved with chair stand performance, but QoL and symptom associations with balance and gait speed were weaker and generally not statistically significant.At time of data analysis, 33 patients (43.4%) were deceased.Decreased survival was associated with baseline severity of fatigue (HR 1.30 per 10-point increase in symptom score, 95% CI 1.12-1.51),dyspnea (HR 1.28, 95% CI 1.11-1.47),and appetite loss (HR 1.27, 95% CI 1.12-1.45).A higher total SPPB score was associated with better survival (HR 0.76, 95% CI 0.61-0.95),while among SPPB components, only chair stand performance was significantly associated with survival (HR 0.67, 95% CI 0.49-0.91).Conclusions: This study confirms the high symptom burden of patients with MPM, with fatigue, dyspnea, insomnia and pain affecting more than half of patients.Our findings additionally show that lower extremity function, specifically measured by ability to perform chair stands, is significantly associated with symptom burden and QoL and may have prognostic significance for MPM patients.Lower extremity function may be a modifiable factor that supportive care interventions can target to improve QoL for patients.

Background: Key transcription factors ASCL1, NEUROD1, POU2F3, YAP1 have been recently reported to characterize uniquely different small cell lung cancer (SCLC) subtypes (SCLC-A; SCLC-N; SCLC-P; SCLC-Y). However, their clinical presence and therapeutic relevance has not yet been widely investigated. Methods: Immunohistochemistry (IHC) was performed on surgically resected specimens of n=386 SCLC patients. Furthermore, large-scale proteomic and in-depth bioinformatical analyses were conducted in n=26 human SCLC cell lines. Standard-of care and targeted agents were used to evaluate distinct therapeutic vulnerabilities in vitro. Results: IHC revealed SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), SCLC-P (POU2F3-dominant) and quadruple-negative SCLC specific subtypes (SCLC-QN) to be present in surgically resected SCLC specimens. Interestingly, SCLC-A subtype significantly correlated with poor and SCLC-P subtype with favorable clinical outcome. Proteomic pathway enrichment analysis identified unique expressional signatures for each SCLC subtype. Cell viability assays demonstrated remarkable sensitivity and resistance differences to standard-of-care chemotherapeutics and targeted agents between distinct SCLC subtypes. Conclusions: Differential expression signatures of four key-transcription factors ASCL1, NEUROD1, POU2F3 and YAP1 are present and clinically relevant in SCLC. Our findings may contribute to a better insight into the biology and therapeutic diversity of SCLC.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy originating from the mesothelial lining of the pleural cavity. Despite the numerous advances in treatment approaches in recent years, the prognosis remains poor with a median overall survival time (OS) ranging from 10 to 22 months. Therefore, the identification of novel non-invasive biomarkers is urgently needed in order to identify patients with a better prognosis and to make the best personalized therapeutic decision. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens.

The tissue distribution and prognostic relevance of subtype‐specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small‐cell lung cancer (SCLC). The expression of subtype‐specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype‐specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC‐A (ASCL1‐dominant), SCLC‐AN (combined ASCL1/NEUROD1), SCLC‐N (NEUROD1‐dominant), and SCLC‐P (POU2F3‐dominant), IHC and cluster analyses identified a quadruple‐negative SCLC subtype (SCLC‐QN). No unique YAP1‐subtype was found. The highest overall survival rates were associated with non‐neuroendocrine subtypes (SCLC‐P and SCLC‐QN) and the lowest with neuroendocrine subtypes (SCLC‐A, SCLC‐N, SCLC‐AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard‐of‐care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype‐specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1‐subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases and is associated with a poor survival rate. SCLC is a tremendously lethal disease characterized by rapid growth and a high propensity to metastasize. Hitherto, platinum-based chemotherapy is one of the key components of the standard first-line therapeutic regimen of SCLC. Despite the known initial chemosensitivity of SCLC, the prospect of therapeutic success is limited by early relapse and acquired resistance in almost all patients.

Small cell lung cancer (SCLC) is a very aggressive type of lung cancer with a poor prognosis due to a high proliferation rate and early metastasis. The current standard-of-care therapeutic treatment, a combination of a platinum-based chemotherapy and etoposide, does not take the four molecular subtypes (SCLC-A, -N, -P and -Y) into account. Oxidative phosphorylation (OXPHOS) is an integral part of cellular metabolism and is frequently altered in cancer. However, not much is known about the role of OXPHOS in SCLC.

Various preclinical studies have stated that members of the MYC transcription factor family (C-myc, L-myc and N-myc) are involved in disease progression and may represent promising therapeutic targets in small cell lung cancer (SCLC). However, there is limited knowledge on the clinical significance of MYC family members in surgically resected SCLC specimens. Our aim was to evaluate the expression and prognostic value of MYC family members in an international cohort of surgically resected SCLC patients.

Background: Pharmacological inhibition of the immune-checkpoint molecule CD47 has shown promising results in preclinical small-cell lung cancer (SCLC) models, whereas anti-programmed death-ligand 1 (PD-L1) inhibitors have been recently implemented in the standard of care of advanced-stage SCLC patients. Nevertheless, the expression pattern, clinical relevance and prognostic implication of both CD47 and PD-L1 are rather controversial in surgically treated SCLC patients.Materials and methods: In total, 104 Caucasian SCLC patients from two Central European thoracic centers were included in this study. CD47 and PD-L1 expression as well as the expression of the four major SCLC molecular subtype markers (ASCL1, NEUROD1, YAP1 and POU2F3) were measured by immunohistochemistry. Expression levels were independently evaluated and statistically correlated with clinicopathological data and survival.Results: Positive CD47 and PD-L1 expressions were seen in 84.6% and 9.6% of the samples, respectively. Meanwhile, the tumor-associated stroma was positive for PD-L1 in 59.6% of the cases. Stromal PD-L1 expression correlated with longer overall survival (OS) (versus PD-L1-negative stroma; median OS was 42 versus 14 months, respectively, P = 0.003) and was confirmed as an independent predictor of favorable outcome upon multivariate analysis (hazard ratio 0.530, 95% confidence interval 0.298-0.943, P = 0.031). Notably, neither CD47 nor PD-L1 presence was related to a distinct molecular SCLC subtype.Conclusion: CD47 shows a remarkably high expression while tumoral PD-L1 expression is generally low in surgically treated SCLC. Importantly, stromal PD-L1 expression may indicate a favorable clinical outcome and serve as a novel prognostic factor in these patients. Additional studies are warranted to further investigate the clinical impact of CD47 and PD-L1 expression in SCLC.

Malignant pleural mesothelioma (MPM) is characterised by aggressive growth and frequent resistance to chemotherapy, poor prognosis for patients and limited therapeutic options. To establish potential new therapy targets, a better understanding of the MPM biology underlying these malignant behaviours is crucial. One potential candidate is the multifunctional oncoprotein YB-1, which is often overexpressed in various cancers and associated with aggressiveness, metastasis and poor outcome.

The fibroblast growth factor (FGF) signaling axis is an important driver of malignant pleural mesothelioma (MPM) growth but the specific contribution of most FGFs has not been explored in detail. In the current study, we focus on FGF5 in MPM and demonstrate its link to poor patient survival, increased tumor cell growth and migration, resistance to cisplatin and increased sensitivity to FGFR inhibitors.

Cell migration is an indispensable function for many cells in multicellular organisms. When deregulated, however, especially in conjunction with the ability to degrade extracellular matrix and invade surrounding tissues, it is a hallmark of malignancy and forms the basis for cancer metastasis. This is often linked to epithelial mesenchymal transition (EMT) which is widely recognized in cancer cell biology to be intricately connected to metastasis, drug resistance and stemness. Multiple extracellular stimuli that induce EMT and cell migration in diverse cellular contexts have been described, nevertheless, a lot still needs to be learned about pathway-specific mechanisms. We have chosen the Ras-mutated A549 lung adenocarcinoma cell line for investigating how the two growth factors EGF and TGFb, which each play fundamental roles in tumor development but activate clearly distinct signaling cascades both stimulate EMT and migration individually and when acting in cooperation. A549 cells were treated with EGF, TGFb or a combination of both in serum-free conditions. Also, inhibitors of downstream pathways were used at sub-lethal concentrations. Changes in cell morphology were determined using ImageJ from microscopy images. Cell migration was assessed by live cell videomicroscopy followed by singe cell tracking. The invasive capacity was determined by a 3D sprouting assay. Expression changes were identified by qPCR and immunoblots. Treatment with TGFb and EGF resulted in cell scattering and distinct changes in cell morphology, which were different for each growth factor. While cells treated with TGFb showed classic EMT-like, elongated morphology, cells exposed to EGF rounded up. Combining both factors resulted in a mixed population. EGF-induced changes could be prevented by Akt but not MAPK inhibitors. Importantly, each growth factor induced a significant increase in cell migration compared to untreated cells and the combination of both factors stimulated migration even further. Interestingly, the increase in migration occurred earlier with EGF than with TGFb, and this was in concordance with increased pERK levels. However, only TGFb was able to induce significantly increased sprouting. Our data describes two independent signaling pathways which both are able to induce cell scattering and cell migration, albeit along different roads. Especially in cancer cells, a better understanding of signaling pathway-dependency of EMT and migration and potential cross talks could lead to more effective antimetastatic therapies.

Malignant pleural mesothelioma (MPM) is an asbestos-related disease with a five-year survival of five percent. Current therapy provides limited success and finding other targetable molecules remains a top priority. We recently identified Y-box binding protein-1 (YB-1) as a significantly overexpressed oncogene with prognostic relevance in MPM. YB-1 is a multifunctional transcription and translation factor of the cold-shock protein family. Using siRNA-mediated knockdown of YB-1 we showed that silencing YB-1 inhibited the proliferation, migration and invasion of four MPM cells by an unknown mechanism. Here we extend this work to examine how YB-1 regulates MPM growth. Functional activity of YB-1 was investigated by siRNA-mediated knockdown in MPM cells followed by TALI apoptosis assays, multi-dimension flow cytometry or live-cell imaging. Transcript expression was determined using reverse transcription qPCR (RT-qPCR) and RNA sequencing (RNA-seq) with poly(A) selection. Following our previous data demonstrating growth inhibition after YB-1 knockdown, we transfected three MPM cell lines with YB-1 siRNA and conducted multi-dimension flow cytometry and TALI apoptosis assays to begin understanding how this growth inhibition was occurring. We found that cells underwent either apoptosis or a G0/G1 cell cycle arrest. Using live-cell imaging and single cell fate mapping we found that each cell line undertook a distinct mechanism of growth inhibition. MSTO cells displayed apoptosis during interphase, VMC23 cells showed no death but underwent a G0/G1 cell cycle arrest, while REN cells did not delay during interphase but entered prolonged aberrant mitosis resulting in mitotic catastrophe and cell death. To examine the interphase arrest in MSTO and VMC23 further we analysed the expression of cyclin D1 and Myc, known cell cycle targets of YB-1, in knockdown samples using RT-qPCR. Transcripts of cyclin D1 and Myc were downregulated in both cell lines in response to reducing YB-1, partially explaining the growth inhibition observed. To further understand the effects of YB-1 inhibition we have undertaken a global analysis of downstream targets and pathways after YB-1 siRNA transfection in all three cell lines using RNA-seq analysis. This project delves into the complex mechanism underlying YB-1-driven MPM proliferation and found it plays a broader role than expected due to its influence over multiple cancer-promoting genes and pathways. Our study significantly extends our understanding of this important protein in MPM, a disease in dire need of actionable targets.

Malignant pleural mesothelioma (MPM) is a devastating disease characterized by aggressive growth and local invasion, poor outcome and limited therapeutic options. YB-1 is a multifunctional oncoprotein, which is often up-regulated in cancer and associated with aggressiveness and poor patient outcome. Also, YB-1 is actively secreted by cells upon various stresses. Besides numerous other functions, YB-1 has been described to stimulate cancer cell migration and invasion via regulation of EMT-related factors such as Snail and Twist. Cells were treated with LPS (20 ng/ml) or grown under hypoxic conditions (1% O2, N2 balanced) for 24 hours. YB-1 levels in supernatants were quantified via western blot. Changes in EMT markers were measured by qPCR. Cell migration and cell cycle was assessed by live cell videomicroscopy followed by manual single cell tracking and analysis using ImageJ and DiPer software, respectively. YB-1 overexpression was achieved by stable transfection with an expression plasmid and confirmed via qPCR and western blot. YB-1 is secreted by MPM cells as well as the immortalised mesothelial cell line Met-5A after exposure to LPS or under hypoxic conditions. When MPM cells as well as primary mesothelial cells derived from pericardial fluid were exposed to soluble YB-1, we observed an upregulation of EMT markers such as SNAIL and TWIST as well as significantly increased migratory capacity. Similar effects were observed in cell lines which overexpress YB-1. YB-1 overexpressing cells migrated at significantly higher speed and covered a larger area. Also, when grown at low density, EMT-like changes in cell morphology as well as scattering was observed. Additionally, while cell divisions occurred at higher frequencies, the duration of the M phase was significantly prolonged. Our data highlight a crucial role of both intracellular and soluble YB-1 in the regulation of migration and invasion, which are key characteristics of MPM. Additionally, YB-1 also interferes with the cell cycle of MPM cells. These findings contribute to a better understanding of the biology of MPM and highlight YB-1’s potential as a therapeutic target.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy and current therapy is essentially palliative. Novel therapy targets are urgently needed. YB-1 is a multifunctional oncoprotein associated with poor patient outcome and is related to increased chemoresistance in tumours including NSCLC. It is widely accepted that YB-1 plays a role in the cell growth of many cancers, and we recently confirmed this in MPM cells. Here, we begin to evaluate YB-1 as a therapeutic target in this disease. YB-1 expression was determined by Western blot in MPM cell lines and their drug resistant sublines. Growth and colony formation assays were conducted after transfection with YB-1- or control-siRNA. These were also carried out in combination with cisplatin, gemcitabine or vinorelbine treatment. Apoptosis was assessed by PI and annexin V staining in YB-1 knockdown MPM cells. Migration of MPM cells was measured using videomicroscopy and manual cell tracking. Luciferase-expressing MPM cells transfected with YB-1- or control-siRNA were injected into female SCID mice (n=10, intra-peritoneal injection). Tumour growth was monitored via luminescence by injecting luciferin (intra-peritoneal injection) and measuring bioluminescence on an In Vitro Imaging System (IVIS) once a week for 4 weeks. Tumour weight determined after humane euthanasia of animals at the termination of the experiment. YB-1-siRNA significantly inhibited the growth of MPM cell lines in vitro and was overexpressed in MPM cells compared to the immortalised mesothelial cell line MeT-5A. Growth of MeT-5A and primary mesothelial cell lines was not affected significantly by YB-1 knockdown. Mice injected with YB-1 knockdown cells displayed significantly lower tumour burden, evidenced by bioluminescence in live mice using IVIS and lower tumour weight after harvest. TALI assays showed an increase in apoptotic cells after YB-1 siRNA transfection in vitro, and cells transfected with siRNA showed sensitisation to cisplatin and vinorelbine. YB-1 was expressed at higher levels, and higher migratory capacity was observed in drug resistant MPM cell lines compared to parental cell lines. These results highlight the importance of YB-1 in MPM biology both in vitro and in vivo. YB-1 knockdown inhibits growth via apoptosis, sensitises MPM cells to commonly prescribed drugs and contributes to a change in behaviour of drug resistant MPM cells. This project serves as a basis for the further investigation of YB-1 as a novel therapeutic target.