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Significance The work by Tesfaye and colleagues defines universal and tumor type–specific features of the p53 tumor suppressor transcriptional network. This study determines a “core” signature of the p53 response across different oncogenic contexts, which defines a universal set of p53 target genes. In addition, this study clarifies the basis for outcome specificity downstream of p53 activation in different oncogenic contexts. We observe that while apoptosis in lymphoma cells is not primarily determined by p53’s transcriptional activity, p53 indirectly promotes senescence in lung adenocarcinoma and sarcoma cells by activating the cis-regulatory long noncoding RNA Pvt1b, which represses Myc levels and its proliferative function., The p53 pathway is a universal tumor suppressor mechanism that limits tumor progression by triggering apoptosis or permanent cell cycle arrest, called senescence. In recent years, efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained traction with the development of a range of small molecules targeting mutant p53. However, knowledge of the downstream mediators of p53 reactivation in different oncogenic contexts has been limited. Here, we utilized a panel of murine cancer cell lines from three distinct tumor types susceptible to alternative outcomes following p53 restoration to define unique and shared p53 transcriptional signatures. While we found that the majority of p53-bound sites and p53-responsive transcripts are tumor-type specific, analysis of shared targets identified a core signature of genes activated by p53 across all contexts. Furthermore, we identified repression of E2F and Myc target genes as a key feature of senescence. Characterization of p53-induced transcripts revealed core and senescence-specific long noncoding RNAs (lncRNAs) that are predominantly chromatin associated and whose production is coupled to cis-regulatory activities. Functional investigation of the contributions of p53-induced lncRNAs to p53-dependent outcomes highlighted Pvt1b, the p53-dependent isoform of Pvt1, as a mediator of p53-dependent senescence via Myc repression. Inhibition of Pvt1b led to decreased activation of senescence markers and increased levels of markers of proliferation. These findings shed light on the core and outcome-specific p53 restoration signatures across different oncogenic contexts and underscore the key role of the p53-Pvt1b-Myc regulatory axis in mediating proliferative arrest.

The identification and functional characterization of long noncoding RNAs (lncRNAs) have suggested a potential source of unexplored cancer therapy targets. The metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was initially identified as a transcript significantly overexpressed in metastatic lung adenocarcinoma (LUAD) and strongly correlated with poor patient outcomes. Despite decades of effort to elucidate Malat1 functions and mechanisms of action in cancer, two key questions have remained unresolved: 1) Is Malat1 overexpression a driver of LUAD progression? and 2) What is the mechanism by which Malat1 overexpression promotes LUAD progression? We utilized CRISPR activation to model Malat1 overexpression in patient-derived LUAD cell lines and in the autochthonous KrasG12D/p53-deficient murine model of LUAD. We observed that Malat1 overexpression cooperates with p53 loss to promote LUAD progression and metastatic dissemination in vivo. Compared to controls, Malat1-overexpressing KrasG12D/p53-deficient tumors appeared poorly differentiated and were characterized by excessive stromal infiltration. Single-cell RNA sequencing of dissociated tumors revealed that Malat1 overexpression triggers pleiotropic immune and stromal responses that favor a metastasis-permissive tumor microenvironment through the secretion of cytokines and metalloproteinases and the recruitment of CAFs and tumor-associated macrophages. Consistent with the in vivo findings, we observed that Malat1 overexpression in vitro does not affect the proliferation but promotes the migration of tumor cells and cancer-associated fibroblasts (CAFs) through secreted factors, including inflammatory cytokines. Importantly, targeting the stabilizing ENE domain of overexpressed Malat1 by two independent approaches in vitro - direct mutagenesis and non-degrading antisense oligonucleotide (ASO) - led to Malat1 destabilization and rescued the increased cellular migration. Similarly, cytokine-targeted blocking antibodies reversed the effects of Malat1 overexpression. In sum, our results represent the first successful efforts to model the increased Malat1 levels observed in human LUAD and to dissect the pro-metastatic molecular consequences of overexpressed Malat1. Our findings indicate that Malat1 is both necessary and sufficient to rewire metastatic pathways and our preliminary data support further efforts to therapeutically target Malat1 and its downstream mediators in metastatic LUAD. Citation Format: Elena Martínez-Terroba, Fernando J. de Miguel, Camila Robles-Oteiza, Katerina Politi, Nadya Dimitrova. Accumulation of the long noncoding RNA Malat1 is a central driver and therapeutic target in metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1540.

Martínez-Terroba and Dimitrova preview work from Athie et al., which demonstrates that the lncRNA ALAL-1 promotes lung cancer cell proliferation and immune evasion., Athie et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201908078) identify ALAL-1, a lncRNA frequently amplified or overexpressed in lung cancer, as an oncogenic driver, capable of promoting the proliferation and altering the immunogenicity of lung cancer cells.

In recent years, the relevance of RNA metabolism has been increasingly recognized in a variety of diseases. Modifications in the levels of RNA-binding proteins elicit changes in the expression of cancer-related genes. Here we evaluate whether SRSF1 regulates the expression of DNA repair genes, and whether this regulation has a relevant role in lung carcinogenesis. An in silico analysis was performed to evaluate the association between the expression of SRSF1 and DNA repair genes. In vitro functional analyses were conducted in SRSF1 or DNA ligase 1 (LIG1)-downregulated non-small cell lung cancer (NSCLC) cell lines. In addition, the prognostic value of LIG1 was evaluated in NSCLC patients by immunohistochemistry. We found a significant correlation between the DNA repair gene LIG1 and SRSF1 in NSCLC cell lines. Moreover, SRSF1 binds to LIG1 mRNA and regulates its expression by increasing its mRNA stability and enhancing its translation in an mTOR-dependent manner. Furthermore, siRNA-mediated LIG1 inhibition reduced proliferation and increased apoptosis of NSCLC cells. Finally, the expression of LIG1 was an independent prognostic factor for NSCLC, as confirmed in a series of 210 patients. These results show that LIG1 is regulated by the oncoprotein SRSF1 and plays a relevant role in lung cancer cell proliferation and progression. LIG1 is associated with poor prognosis in non-small lung cancer patients.

Each of the pathological stages (I-IIIa) of surgically resected non-small-cell lung cancer has hidden biological heterogeneity, manifested as heterogeneous outcomes within each stage. Thus, the finding of robust and precise molecular classifiers with which to assess individual patient risk is an unmet medical need. Here, we identified and validated the clinical utility of a new prognostic signature based on three proteins (BRCA1, QKI, and SLC2A1) to stratify early-stage lung adenocarcinoma patients according to their risk of recurrence or death. Patients were staged according to the new International Association for the Study of Lung Cancer (IASLC) staging criteria (8th edition, 2018). A test cohort (n = 239) was used to assess the value of this new prognostic index (PI) based on the three proteins. The prognostic signature was developed by Cox regression with the use of stringent statistical criteria (TRIPOD: Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis). The model resulted in a highly significant predictor of 5-year outcome for disease-free survival (p < 0.001) and overall survival (p < 0.001). The prognostic ability of the model was externally validated in an independent multi-institutional cohort of patients (n = 114, p = 0.021). We also demonstrated that this molecular classifier adds relevant information to the gold standard TNM-based pathological staging, with a highly significant improvement of the likelihood ratio. We subsequently developed a combined PI including both the molecular and the pathological data that improved the risk stratification in both cohorts (p ≤ 0.001). Moreover, the signature may help to select stage I-IIA patients who might benefit from adjuvant chemotherapy. In summary, this protein-based signature accurately identifies those patients with a high risk of recurrence and death, and adds further prognostic information to the TNM-based clinical staging, even when the new IASLC 8th edition staging criteria are applied. More importantly, it may be a valuable tool for selecting patients for adjuvant therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood.To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site.Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed.High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16.Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.

Summary The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.

The p53 pathway is a key tumor suppressor mechanism that stalls cell cycle progression in response to stress. It has been proposed that p53 suppresses proliferation in part by downregulating the Myc oncogene but the mechanism has remained unclear. Here, we show that p53 directly represses Myc through a long noncoding RNA expressed from the Pvt1 locus. Pvt1b, a p53-dependent RNA isoform, is rapidly induced upon genotoxic and oncogenic stress and accumulates at its site of transcription in foci that exhibit properties of liquid-phase condensates. Pvt1b uses long-range chromatin interactions between Pvt1b and Myc to locally suppress Myc in an RNA-dependent manner. Loss of p53-dependent Pvt1b induction leads to increased cellular proliferation in vitro and enhanced tumor growth in a mouse model of lung adenocarcinoma in vivo. We propose that Pvt1b-mediated Myc repression contributes to the p53 tumor suppressive network to limit cancer growth.

IntroductionPrognostic biomarkers have been very elusive in the lung squamous cell carcinoma (SCC) and none is currently being used in the clinical setting. We aimed to identify and validate the clinical utility of a protein-based prognostic signature to stratify patients with early lung SCC according to their risk of recurrence or death.MethodsPatients were staged following the new International Association for the Study of Lung Cancer (IASLC) staging criteria (eighth edition, 2018). Three independent retrospective cohorts of 117, 96 and 105 patients with lung SCC were analysed to develop and validate a prognostic signature based on immunohistochemistry for five proteins.ResultsWe identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (pConclusionWe have identified and validated a clinically feasible protein-based prognostic model that complements the updated TNM system allowing more accurate risk stratification. This signature may be used as an advantageous tool to improve the clinical management of the patients, allowing the reduction of lung SCC mortality through a more accurate knowledge of the patient’s potential outcome.

BACKGROUND:Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology. MATERIAL AND METHODS:Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor. RESULTS:High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P