Laser ablation-inductively coupled plasma-mass spectrometry analysis reveals differences in chemotherapeutic drug distribution in surgically resected pleural mesothelioma.

AIMS: Pleural mesothelioma (PM) is a highly aggressive thoracic tumour with poor prognosis. Although reduced tissue drug accumulation is one of the key features of platinum (Pt) resistance, little is known about Pt distribution in human PM. METHODS: We assessed Pt levels of blood samples and surgically resected specimens from 25 PM patients who had received neoadjuvant Pt-based chemotherapy (CHT). Pt levels and tissue distributions were measured by laser ablation-inductively coupled plasma-mass spectrometry and correlated with clinicopathological features. RESULTS: In surgically resected PM specimens, mean Pt levels of nontumourous (fibrotic) areas were significantly higher (vs tumourous regions, P = 0.0031). No major heterogeneity of Pt distribution was seen within the tumourous areas. Pt levels correlated neither with the microvessel area nor with apoptosis rate in the tumourous or nontumourous regions. A significant positive correlation was found between serum and both full tissue section and tumourous area mean Pt levels (r = 0.532, P = 0.006, 95% confidence interval [95% CI] 0.161-0.771 and r = 0.415, P = 0.039, 95% CI 0.011-0.702, respectively). Furthermore, a significant negative correlation was detected between serum Pt concentrations and elapsed time from the last cycle of CHT (r = -0.474, P = 0.017, 95% CI -0.738--0.084). Serum Pt levels correlated negatively with overall survival (OS) (P = 0.029). CONCLUSIONS: There are major differences in drug distribution between tumourous and nontumourous areas of PM specimens. Serum Pt levels significantly correlate with full section and tumourous area average Pt levels, elapsed time from the last CHT cycle, and OS. Further studies investigating clinicopathological factors that modulate tissue Pt concentration and distribution are warranted.

Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer.

BACKGROUND: No targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies. METHODS: Expression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids. RESULTS: The highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction. CONCLUSIONS: The current study reveals the subtype specificity of BCL-2 expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance. METHODS: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues. RESULTS: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO). CONCLUSIONS: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

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.

Molecular profiles of small cell lung cancer subtypes: therapeutic implications.

Small cell lung cancer (SCLC; accounting for approximately 13%-15% of all lung cancers) is an exceptionally lethal malignancy characterized by rapid doubling time and high propensity to metastasize. In contrast to the increasingly personalized therapies in other types of lung cancer, SCLC is still regarded as a homogeneous disease and the prognosis of SCLC patients remains poor. Recently, however, substantial progress has been made in our understanding of SCLC biology. Advances in genomics and development of new preclinical models have facilitated insights into the intratumoral heterogeneity and specific genetic alterations of this disease. This worldwide resurgence of studies on SCLC has ultimately led to the development of novel subtype-specific classifications primarily based on the neuroendocrine features and distinct molecular profiles of SCLC. Importantly, these biologically distinct subtypes might define unique therapeutic vulnerabilities. Herein, we summarize the current knowledge on the molecular profiles of SCLC subtypes with a focus on their potential clinical implications.

[Heterogeneity of small cell lung cancer: biological and clinicopathological implications]. / A kissejtes tüdorák heterogenitásának biológiai és klinikopatológiai jelentosége.

Small cell lung cancer (SCLC; comprising approximately 14% of all lung cancer cases in Hungary) is an aggressive tumor type characterized by rapid growth and early metastasis. Although SCLC is a particularly malignant form of cancer, targeted therapies in its treatment have remained largely unsuccessful and thus there were no major therapeutic advances in the last three decades. SCLC was once considered a molecularly homogeneous malignancy. However, recent analyses led to the classification of neuroendocrine and molecular subtypes, based on the dominant expression of one of the following four transcriptional regulator genes: ASCL1, NEUROD1, YAP1 and POU2F3. Because these genetically and biologically distinct subtypes might contribute to therapeutic resistance, the better understanding of their biological and clinicopathological characteristics may help in the development of more effective SCLC therapies.

The FAK inhibitor BI 853520 inhibits spheroid formation and orthotopic tumor growth in malignant pleural mesothelioma.

No tyrosine kinase inhibitors are approved for malignant pleural mesothelioma (MPM). Preclinical studies identified focal adhesion kinase (FAK) as a target in MPM. Accordingly, we assessed the novel, highly selective FAK inhibitor (BI 853520) in 2D and 3D cultures and in vivo. IC50 values were measured by adherent cell viability assay. Cell migration and 3D growth were quantified by video microscopy and spheroid formation, respectively. Phosphorylation of FAK, Akt, S6, and Erk was measured by immunoblot. The mRNA expression of the putative tumor stem cell markers SOX2, Nanog, CD44, ALDH1, c-myc, and Oct4 was analyzed by qPCR. Cell proliferation, apoptosis, and tumor tissue microvessel density (MVD) were investigated in orthotopic MPM xenografts. In all 12 MPM cell lines, IC50 exceeded 5 µM and loss of NF2 did not correlate with sensitivity. No synergism was found with cisplatin in adherent cells. BI 853520 decreased migration in 3 out of 4 cell lines. FAK phosphorylation was reduced upon treatment but activation of Erk, Akt, or S6 remained unaffected. Nevertheless, BI 853520 inhibited spheroid growth and significantly reduced tumor weight, cell proliferation, and MVD in vivo. BI 853520 has limited effect in adherent cultures but demonstrates potent activity in spheroids and in orthotopic tumors in vivo. Based on our findings, further studies are warranted to explore the clinical utility of BI 853520 in human MPM. KEY MESSAGES: Response to FAK inhibition in MPM is independent of NF2 expression or histotype. FAK inhibition strongly interfered with MPM spheroid formation. BI 853520 has been shown to exert anti-tumor effect in MPM.

Nintedanib Is Active in Malignant Pleural Mesothelioma Cell Models and Inhibits Angiogenesis and Tumor Growth In Vivo.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive thoracic tumor type with limited treatment options and poor prognosis. The angiokinase inhibitor nintedanib has shown promising activity in the LUME-Meso phase II MPM trial and thus is currently being evaluated in the confirmatory LUME-Meso phase III trial. However, the anti-MPM potential of nintedanib has not been studied in the preclinical setting.Experimental Design: We have examined the antineoplastic activity of nintedanib in various in vitro and in vivo models of human MPM.Results: Nintedanib's target receptors were (co)expressed in all the 20 investigated human MPM cell lines. Nintedanib inhibited MPM cell growth in both short- and long-term viability assays. Reduced MPM cell proliferation and migration and the inhibition of Erk1/2 phosphorylation were also observed upon nintedanib treatment in vitro Additive effects on cell viability were detected when nintedanib was combined with cisplatin, a drug routinely used for systemic MPM therapy. In an orthotopic mouse model of human MPM, survival of animals receiving nintedanib per os showed a favorable trend, but no significant benefit. Nintedanib significantly reduced tumor burden and vascularization and prolonged the survival of mice when it was administered intraperitoneally. Importantly, unlike bevacizumab, nintedanib demonstrated significant in vivo antivascular and antitumor potential independently of baseline VEGF-A levels.Conclusions: Nintedanib exerts significant antitumor activity in MPM both in vitro and in vivo These data provide preclinical support for the concept of LUME-Meso trials evaluating nintedanib in patients with unresectable MPM. Clin Cancer Res; 24(15); 3729-40. ©2018 AACR.

The role of p38 signaling and poly(ADP-ribosyl)ation-induced metabolic collapse in the osteogenic differentiation-coupled cell death pathway.

Osteogenic differentiation is a multistep process regulated by a diverse set of morphogenic and transcription factors. Previously we identified endogenous hydrogen peroxide-induced poly(ADP-ribose) polymerase-1 (PARP1) activation as a mediator of osteodifferentiation and associated cell death. Here we set out to investigate whether or not activation of PARP1 is dependent on DNA breaks and how PARP1 mediates cell death during osteodifferentiation of mesenchymal stem cells and SAOS-2 cells. Here we show that the MAP kinases p38, JNK, and ERK1/2 become activated during the differentiation process. However, only p38 activation depended both on hydrogen peroxide production and on PARP1 activation as the hydrogen peroxide decomposing enzyme catalase, the PARP inhibitor PJ34, and the silencing of PARP1 suppressed p38 activation. Inhibition of p38 suppressed cell death and inhibited osteogenic differentiation (calcium deposition, alkaline phosphatase activity, and marker gene expression) providing further support for the close coupling of osteodifferentiation and cell death. Metabolic collapse appears to be central in the hydrogen peroxide-PARP1-p38 pathway as silencing PARP1 or inhibition of p38 prevented differentiation-associated loss of cellular NAD, inhibition of mitochondrial respiration, and glycolytic activity. We also provide evidence that endogenous hydrogen peroxide produced by the differentiating cells is sufficient to cause detectable DNA breakage. Moreover, p38 translocates from the cytoplasm to the nucleus where it interacts and colocalizes with PARP1 as detected by immunoprecipitation and immunofluorescence, respectively. In summary, hydrogen peroxide-induced PARP1 activation leads to p38 activation and this pathway is required both for the successful completion of the differentiation process and for the associated cell death.