p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

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 transcriptional response across tumor types reveals core and senescence-specific signatures modulated by long noncoding RNAs.

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.

5 protein-based signature for resectable lung squamous cell carcinoma improves the prognostic performance of the TNM staging.

INTRODUCTION: Prognostic 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. METHODS: Patients 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. RESULTS: We identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (p<0.001; HR=4.06, 95% CI 2.18 to 7.56) and overall survival (OS) (p=0.004; HR=2.38, 95% CI 1.32 to 4.31). The prognostic capability of PI was confirmed in an external multi-institutional cohort for DFS (p=0.042; HR=2.01, 95% CI 1.03 to 3.94) and for OS (p=0.031; HR=2.29, 95% CI 1.08 to 4.86). Moreover, PI added complementary information to the newly established IASLC TNM 8th edition staging system. A combined prognostic model including both molecular and anatomical (TNM) criteria improved the risk stratification in both cohorts (p<0.05). CONCLUSION: We 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.

A novel protein-based prognostic signature improves risk stratification to guide clinical management in early-stage lung adenocarcinoma 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.

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

RATIONALE: 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. OBJECTIVES: 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. METHODS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

The oncogenic RNA-binding protein SRSF1 regulates LIG1 in non-small cell lung cancer.

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.

Cereblon-based Bifunctional Degrader of SOS1, BTX-6654, Targets Multiple KRAS Mutations and Inhibits Tumor Growth.

Mutations within the oncogene KRAS drive an estimated 25% of all cancers. Only allele-specific KRAS G12C inhibitors are currently available and are associated with the emergence of acquired resistance, partly due to upstream pathway reactivation. Given its upstream role in the activation of KRAS, son of sevenless homolog 1 (SOS1), has emerged as an attractive therapeutic target. Agents that target SOS1 for degradation could represent a potential pan-KRAS modality that may be capable of circumventing certain acquired resistance mechanisms. Here, we report the development of two SOS1 cereblon-based bifunctional degraders, BTX-6654 and BTX-7312, cereblon-based bifunctional SOS1 degraders. Both compounds exhibited potent target-dependent and -specific SOS1 degradation. BTX-6654 and BTX-7312 reduced downstream signaling markers, pERK and pS6, and displayed antiproliferative activity in cells harboring various KRAS mutations. In two KRAS G12C xenograft models, BTX-6654 degraded SOS1 in a dose-dependent manner correlating with tumor growth inhibition, additionally exhibiting synergy with KRAS and MEK inhibitors. Altogether, BTX-6654 provided preclinical proof of concept for single-agent and combination use of bifunctional SOS1 degraders in KRAS-driven cancers.

Overexpression of Malat1 drives metastasis through inflammatory reprogramming of the tumor microenvironment.

Expression of the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) correlates with tumor progression and metastasis in many tumor types. However, the impact and mechanism of action by which MALAT1 promotes metastatic disease remain elusive. Here, we used CRISPR activation (CRISPRa) to overexpress MALAT1/Malat1 in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. Malat1 overexpression was sufficient to promote the progression of LUAD to metastatic disease in mice. Overexpression of MALAT1/Malat1 enhanced cell mobility and promoted the recruitment of protumorigenic macrophages to the tumor microenvironment through paracrine secretion of CCL2/Ccl2. Ccl2 up-regulation was the result of increased global chromatin accessibility upon Malat1 overexpression. Macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

Long noncoding RNA amplified in lung cancer rewires cancer pathways.

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.

A large-scale analysis of alternative splicing reveals a key role of QKI in lung cancer.

Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.

Expression of sirtuin 1 and 2 is associated with poor prognosis in non-small cell lung cancer patients.

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<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC. CONCLUSIONS: Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.