Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition

IκB kinase α is required for development and progression of KRAS-mutant lung adenocarcinoma.

Although oncogenic activation of NF-κΒ has been identified in various tumors, the NF-κΒ-activating kinases (IKKs) responsible for this are elusive. Here we report an actionable requirement for IKKα in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. For this, we mapped NF-κΒ activation in the lungs during chemical and genetic adenocarcinoma development and found two distinct early and late activation phases, which were characterized by nuclear translocation of RelB, ΙκΒβ, and /or IKKα in tumor-initiated cells. We further used conditional IKKα and ΙΚΚβ-deleted mice to identify that IKKα is a cardinal tumor promoter in KRAS-mutant lung adenocarcinoma. We also co-expressed IKKα or ΙΚΚβ with wild-type or mutant KRAS in benign cells, and show that IKKα cooperates with mutant KRAS for tumor induction. Finally, we show that IKKα is highly expressed in human lung adenocarcinomas, and that a dual IKKα/β inhibitor delivers superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results position IKKα as a target against KRAS-mutant lung adenocarcinoma and provide proof-of-principle data for future targeting of KRAS-IKKα addiction in the disease.Παρ’ όλο που η ογκογόνος ενεργοποίηση του πυρηνικού παράγοντα NF-κΒ έχει ανιχνευθεί σε διάφορους όγκους, ο ρόλος των υπεύθυνων κινασών-ενεργοποιητών του NF-κΒ (IKKs) παραμένουν αχαρτογράφητος. Εδώ περιγράφουμε πως η κινάση ΙΚΚα είναι απαραίτητη για την ανάπτυξη αδενοκαρκινώματος πνεύμονα επαγόμενου μέσω άμεσης αναπνευστικής επιθηλιακής έκφρασης ογκογόνου KRASG12D. Για το σκοπό αυτό, ιχνηλατήσαμε την ενεργότητα του NF-κΒ στους πνεύμονες κατά τη διάρκεια γενετικά επαγόμενης καρκινογένεσης και βρήκαμε δύο διακριτές πρώιμες και όψιμες φάσεις ενεργοποίησής του, οι οποίες χαρακτηρίζονται από πυρηνική μετατόπιση της IKKα στα καρκινικά κύτταρα. Στη συνέχεια χρησιμοποιήσαμε υπό συνθήκες ανεπαρκείς στα γονίδια των IKKα και ΙΚΚβ ποντικούς για να ανακαλύψουμε πως η IKKα είναι πιο σημαντική ακόμη και από την IKKβ για την ανάπτυξη αδενοκαρκινώματος. Τέλος δείχνουμε πως η θεραπευτική στόχευση της IKKα με χρήση διπλού αναστολέα των IKK παρέχει υψηλότερου βαθμού προστασία έναντι της ανάπτυξης KRAS-μεταλλαγμένου αδενοκαρκινώματος πνεύμονα σε σύγκριση με τον ειδικό αναστολέα της IKKβ. Τα αποτελέσματα αυτά καθιστούν την IKKα σημαντικό θεραπευτικό στόχο έναντι του αδενοκαρκινώματος πνεύμονα που φέρει μεταλλάξεις του KRAS και παρέχουν πρωταρχικά δεδομένα που υποστηρίζουν μελλοντική στόχευση της εξάρτησης KRAS-IKKα ενάντια σε αυτή την επάρατη νόσο

Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC

Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.

<p><b>(A)</b> Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. <b>(B)</b> NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. <b>(C)</b> Representative images of PCNA-stained NSCLC subtype samples. <b>(D)</b> Nuclear co-localization of PCNA immunoreactivity with <i>Rel</i>B (arrows), but not with <i>Rel</i>A, was identified using dual immunostaining of samples of 10 patients (representative images shown).</p

Immunohistochemical detection of NF-κB in mouse models of NSCLC.

<p>NF-κB subunit expression was assessed by immunohistochemistry in urethane-induced mouse lung adenomas <b>(A and C)</b> and mutant <i>KRAS</i>-induced lung adenocarcinomas <b>(B and D)</b>. <b>(A, B)</b> Representative images. <b>(C, D)</b> Overall scoring of NF-κB subunit expression levels from four mice per group. Data presented as mean ± SD. ** and ***: P < 0.01, and P < 0.001 for the indicated color-coded subunit compared with normal bronchial and alveolar epithelium by two-way ANOVA followed by Bonferroni post-tests. Non-significant comparisons are not indicated.</p

NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.

<p><b>(A)</b> Representative images. <b>(B, D)</b> Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C, E)</b> Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(F)</b> Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(G)</b> Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.</p

Immunohistochemical detection of NF-κB subunits in NSCLC, juxta-tumoral normal lung structures and preneoplastic lesions.

<p><b>(A)</b> Representative images. Images in red frames representatively display differential NF-κB subunit expression in tumor and intratumoral stroma areas. <b>(B)</b> Overall scoring of NF-κB subunit expression levels. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, * and ***: P > 0.05, P < 0.05, and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C)</b> Co-expression matrixes of categorical NF-κB subunit expression levels. For this, NF-κB scores from (B) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 by χ² tests followed by Fisher’s exact tests.</p

Association of NF-κB expression with clinical and pathologic parameters in 77 patients with NSCLC.

<p><b>(A)</b> NF-κB expression levels subdivided by clinical and pathological parameters. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, *, and **: P > 0.05, P < 0.05, and P < 0.0501 for indicated comparisons by Wilcoxon signed rank tests or Kruskal-Wallis tests followed by Dunn’s post-tests, for two or multiple comparison groups, respectively. <b>(B)</b> Results of binary logistic regression analyses using NF-κB subunit expression scores as the input (independent variables) and dichotomized clinical and pathologic parameters as the output (dependent variables). RR, risk ratios; CI, confidence intervals; P, probability values.</p

Schematic illustration of the main findings of the present study.

<p>NF-κB subunit expression levels in tumor and stroma cells of 77 patients with NSCLC are indicated by relative font size. Arrows indicate possible associations of <i>Rel</i> protein expression levels in NSCLC tumor cells with tumor-associated inflammation and cellular proliferation.</p