A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma

Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)–myosin IIA–containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a–dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8⁺ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.     

Transformation of non-small cell lung cancer into small cell lung cancer in a patient with advanced lung cancer: a case report

Targeted therapy in patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) often fails because of drug resistance. Here, we report a 57-year-old male patient with stage IV small cell lung cancer (SCLC) transformation during targeted therapy. Chest computerized tomography (CT), hematoxylin and eosin histological examination, immunohistochemistry, allele refractory mutation system‐based quantitative polymerase chain reaction analysis of EGFR point mutations, and next-generation sequencing were performed for diagnosis and therapeutic efficacy evaluation. A combination of chest CT, histological examination, and immunohistochemistry confirmed the initial NSCLC diagnosis. Next-generation sequencing detected only EGFR exon 19 deletion (ex19del) before treatment and later identified EGFR exon20p.T790M point mutation, EGFR amplification, myc proto-oncogene (MYC) amplification, retinoblastoma 1 (RB1) mutation, and tumor protein 53 (TP53) mutation. Histology and immunohistochemistry revealed transformation from NSCLC to SCLC during treatment, which eventually returned to NSCLC. Drug resistance to targeted therapy for patients with NSCLC frequently occurs because of EGFR exon20p.T790M point mutation, TP53 mutation, RB1 mutation, and MYC amplification. These mutations are also the major determining factors of NSCLC outcomes. Therefore, next-generation sequencing should be performed to confirm drug efficacy during targeted therapy for NSCLC.     

PRP19 upregulation inhibits cell proliferation in lung adenocarcinomas by p21-mediated induction of cell cycle arrest

Precursor messenger RNA processing factor 19 (PRP19) is known to be a critical component of the eukaryotic spliceosomal machinery and DNA damage repair system, the deregulation of which leads to many disease conditions. In many human cancers, PRP19 expression is upregulated, but its functional significance and corresponding underlying mechanisms remain to be addressed. Focusing on lung carcinomas, PRP19 upregulation was achieved by plasmid transfection into A549 adenocarcinoma cells. The transfected cells were then subjected to several in vitro and in vivo assays following in situ assessment of the protein in paired clinical lung tissues. We report that PRP19 expression is elevated in lung carcinoma tissues compared to non-tumor tissues. Following its upregulation, PRP19 repressed cell proliferation and tumor growth by upregulating the expression of the cell cycle arrest protein p21.     

Uncovering the dosage-dependent roles of Arid1a in gastric tumorigenesis for combinatorial drug therapy

Gastric cancer (GC) is one of the most common deadly cancers in the world. Although patient genomic data have identified AT-rich interaction domain 1A (ARID1A), a key chromatin remodeling complex subunit, as the second most frequently mutated gene after TP53, its in vivo role and relationship to TP53 in gastric tumorigenesis remains unclear. Establishing a novel mouse model that reflects the ARID1A heterozygous mutations found in the majority of human GC cases, we demonstrated that Arid1a heterozygosity facilitates tumor progression through a global loss of enhancers and subsequent suppression of the p53 and apoptosis pathways. Moreover, mouse genetic and single-cell analyses demonstrated that the homozygous deletion of Arid1a confers a competitive disadvantage through the activation of the p53 pathway, highlighting its distinct dosage-dependent roles. Using this unique vulnerability of Arid1a mutated GC cells, our combined treatment with the epigenetic inhibitor, TP064, and the p53 agonist, Nutlin-3, inhibited growth of Arid1a heterozygous tumor organoids, providing a novel therapeutic option for GC.     

Mutant p53–associated myosin-X upregulation promotes breast cancer invasion and metastasis

Mutations of the tumor suppressor TP53 are present in many forms of human cancer and are associated with increased tumor cell invasion and metastasis. Several mechanisms have been identified for promoting dissemination of cancer cells with TP53 mutations, including increased targeting of integrins to the plasma membrane. Here, we demonstrate a role for the filopodia-inducing motor protein Myosin-X (Myo10) in mutant p53–driven cancer invasion. Analysis of gene expression profiles from 2 breast cancer data sets revealed that MYO10 was highly expressed in aggressive cancer subtypes. Myo10 was required for breast cancer cell invasion and dissemination in multiple cancer cell lines and murine models of cancer metastasis. Evaluation of a Myo10 mutant without the integrin-binding domain revealed that the ability of Myo10 to transport β₁ integrins to the filopodia tip is required for invasion. Introduction of mutant p53 promoted Myo10 expression in cancer cells and pancreatic ductal adenocarcinoma in mice, whereas suppression of endogenous mutant p53 attenuated Myo10 levels and cell invasion. In clinical breast carcinomas, Myo10 was predominantly expressed at the invasive edges and correlated with the presence of TP53 mutations and poor prognosis. These data indicate that Myo10 upregulation in mutant p53–driven cancers is necessary for invasion and that plasma-membrane protrusions, such as filopodia, may serve as specialized metastatic engines.     

Rb deletion in mouse mammary progenitors induces luminal-B or basal-like/EMT tumor subtypes depending on p53 status

Breast cancer is a highly heterogeneous disease, with several different subtypes being characterized by distinct histology, gene expression patterns, and genetic alterations. The tumor suppressor gene retinoblastoma 1 (RB1) is frequently lost in both luminal-B and triple-negative tumor (TNT; i.e., estrogen receptor–, progesterone receptor–, and human epidermal growth factor receptor 2–negative) breast cancer subtypes. However, a causal role for RB1 loss in different subtypes remains undefined. Here we report that deletion of Rb alone or together with its relative p107 in mouse mammary stem/bipotent progenitor cells induced focal acinar hyperplasia with squamous metaplasia. These lesions progressed into histologically diverse, transplantable mammary tumors with features of either luminal-B or TNT subtypes. The TNTs included basal-like tumors as well as tumors that exhibited epithelial-to-mesenchymal transition (EMT). The EMT-type tumors and a subset of the basal-like tumors, but not luminal-B–like tumors, expressed mutant forms of the tumor suppressor p53. Accordingly, targeted deletion of both Rb and p53 in stem/bipotent progenitors led to histologically uniform, aggressive, EMT-type tumors. Reintroduction of Rb into these tumor cells suppressed growth in vitro and tumor formation in vivo. These results establish a causal role for Rb loss in breast cancer in mice and demonstrate that cooperating oncogenic events, such as mutations in p53, dictate tumor subtype after Rb inactivation.     

Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis

Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine Kras^G12D-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of Kras^G12D-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers.     

Serum anti-p53 antibody detection in carcinomas and the predictive values of serum p53 antibodies, carcino-embryonic antigen and carbohydrate antigen 12-5 in the neoadjuvant chemotherapy treatment for III stage non-small cell lung cancer patients

BackgroundThe serum p53 antibody (s-p53 Ab) is a valuable prognostic factor for carcinomas, but its common detection method, based on enzyme linked immunosorbent assay (ELISA), needs to be improved due to low sensitivity. Although neoadjuvant chemotherapy (NACT) is widely used in the treatment of non-small cell lung cancer (NSCLC) in China, forecasting chemoresistance is still a pressing problem.MethodsHybrid phage and wild-type p53 protein (wt p53 protein) were produced before the establishment of phage-ELISA and p53-ELISA. S-p53 Abs of 829 patients with various types of cancer was detected by a double ELISA system. 47 ΙΙΙ stage NSCLC patients treated with mitomycin, vindesine and cisplatin (MCV)-based NACT were chosen for s-p53 Abs, carcino-embryonic antigen (CEA) and carbohydrate antigen (CA) 12-5 predictive value analysis.ResultsThrough the combination of p53-ELISA and phage-ELISA (p53-phage ELISA), the sensitivity of s-p53 Abs in lung, breast, colorectal, gastric, esophageal, liver and ovarian cancer increased to 39.0%, 33.3%, 41.7%, 32.1%, 30.9%, 23.1% and 43.2% respectively. S-p53 Abs proved to correlate with nodal involvement, TNM stage, histological type (in lung cancer) or tumor size (in gastric cancer). As for the 47 ΙΙΙ stage NSCLC treated with NACT, s-p53 Abs and CA12-5 remarkably decreased after NACT treatment (P = 0.034 and P = 0.007) and pre-NACT low s-p53 Abs correlated with high objective chemoresponse rate (P = 0.016).Conclusionsp53-phage ELISA system has an edge over single p53-ELISA. S-p53 Abs level correlates with cancer patients' clinicalpathological parameters and can predict the chemoresponse of ΙΙΙ stage NSCLC patients during MCV-based NACT treatment.     

利用组织芯片技术观察p53及其同系物p63在恶性肿瘤中的表达

目的探讨p53和p63在部分肿瘤发生中的作用,为其人群大规模筛查、早期诊断及预后评估提供依据。方法选取病理科档案材料中食管鳞癌、胃腺癌、大肠腺癌、膀胱移行细胞癌、肺癌、肝细胞性癌、乳腺浸润导管癌、肾透明细胞癌、卵巢腺样囊腺癌和胰腺癌石蜡包埋组织各6例及相应正常组织各3例。肿瘤标本先观察苏木精-伊红(HE)切片以确定肿瘤部位,采用组织芯片仪及手工法制作180点列阵组织芯片,进行p53和p63抗体免疫组化染色。光镜观察p53、p63阳性细胞在各组的分布,并进行对比分析。结果p53表达类型:(1)正常组织p53阳性细胞数很少;(2)3例食管癌、3例胃癌、2例大肠癌、1例肺癌、1例乳腺癌、2例卵巢癌的绝大多数肿瘤细胞呈强阳性核染色反应,癌旁组织细胞和间质细胞阴性,1例膀胱癌中有少量阳性肿瘤细胞散在分布于阴性肿瘤细胞中。p63表达类型:(1)正常食道复层扁平上皮基底层细胞呈核染色,p63阳性细胞散在分布于棘细胞层;(2)6例食道癌、6例膀胱癌和4例肺鳞癌均为强阳性反应,大多数肿瘤细胞呈强核染色,癌旁组织细胞和间质细胞p63阴性;两例肺腺癌p63阴性;(3)6例胃癌中的4例、6例大肠癌中的3例、6例乳腺浸润导管癌中的2例、6例肝细胞性癌中的1例可见较强的肿瘤细胞胞质染色。其中3例食管癌、3例胃癌、1例大肠癌、1例肺     

周围型肺癌76例患者的CT表现、血清肿瘤标记物及Ki-67、p53的蛋白表达的相关性研究

目的探讨周围型肺癌患者的CT表现、血清肿瘤标记物及Ki-67、p53的蛋白表达的相关性。方法回顾性分析2008年1月至2011年12月周围型肺癌76例患者的CT和血清肿瘤标记物资料,评价不同病理类型的周围型肺癌的CT表现,Ki-67、p53蛋白表达与血清癌胚抗原（CEA）、神经元特异性烯醇化酶（NSE）、细胞角蛋白19片段的关系。结果不同病理类型的周围型肺癌,其CT表现毛刺征、分叶征、胸膜凹陷征的阳性率差异有统计学意义（P〈0.05）;鳞癌的Ki-67水平高于腺癌。结论血清肿瘤标志物联合影像检测能够提高肿瘤的检出率。Ki-67、p53的表达程度能反映肿瘤的恶性程度。     

Guanylate‐binding protein 1 correlates with advanced tumor features,and serves as a prognostic biomarker for worse survival in lung adenocarcinoma patients

ObjectiveGuanylate‐binding protein 1 (GBP1) is reported to promote tumor progression and treatment resistance in lung cancer, and presents as a prognostic biomarker in several solid tumors. However, the related research of GBP1 in clinical management of lung adenocarcinoma is still lacking. Therefore, the present study aimed to detect the clinical role of GBP1 in lung adenocarcinoma.MethodsThe clinical data of 221 lung adenocarcinoma patients were retrospectively analyzed, and then, their tumor tissue specimens and paired adjacent tissue specimens were retrieved for GBP1 detection via immunohistochemistry (IHC) assay.ResultsGBP1 expression was upregulated in tumor tissues compared with adjacent tissues (P < .001). Moreover, high tumor GBP1 expression was associated with larger tumor size (P = .030), positive lymph node (LYN) metastasis (P = .001), advanced TNM stage (P = .001), and abnormal preoperative carcinoembryonic antigen (CEA) level (P = .026). Furthermore, tumor GBP1 high expression was correlated with reduced disease‐free survival (DFS) and overall survival (OS), and was of independent value in predicting worse DFS and OS. Additionally, data analysis of 1144 lung cancer patients derived from KMplot database (www.kmplot.com) further verified that GBP1 expression was negatively correlated with OS (P = .009).ConclusionGBP1 correlates with advanced tumor features and worse survival profiles, suggesting its value to be a prognostic biomarker in management of lung adenocarcinoma.     

Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors

Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1^–/– background. We found that oncogenic Kras–induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.     

Therapeutic Effects of MicroRNA-582-5p and -3p on the Inhibition of Bladder Cancer Progression

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.     

Using Dual Fluorescence Reporting Genes to Establish an <Emphasis Type="Italic">In Vivo</Emphasis> Imaging Model of Orthotopic Lung Adenocarcinoma in Mice

Purpose

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy.

Procedures

A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system.

Results

For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells.

Conclusions

We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.