Upregulation of mRNA Expression of ADGRD1/GPR133 and ADGRG7/GPR128 in SARS-CoV-2-Infected Lung Adenocarcinoma Calu-3 Cells.

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Cathepsin B is a potential therapeutic target for coronavirus disease 2019 patients with lung adenocarcinoma.

Coronavirus disease 2019 (COVID-19) was declared a serious global public health emergency. Hospitalization and mortality rates of lung cancer patients diagnosed with COVID-19 are higher than those of patients presenting with other cancers. However, the reasons for the outcomes being disproportionately severe in lung adenocarcinoma (LUAD) patients with COVID-19 remain elusive. The present study aimed to identify the possible causes for disproportionately severe COVID-19 outcomes in LUAD patients and determine a therapeutic target for COVID-19 patients with LUAD. We used publicly available data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and various bioinformatics tools to identify and analyze the genes implicated in SARS-CoV-2 infection in LUAD patients. Upregulation of the SARS-CoV-2 infection-related molecules dipeptidyl peptidase 4, basigin, cathepsin B (CTSB), methylenetetrahydrofolate dehydrogenase, and peptidylprolyl isomerase B rather than angiotensin-converting enzyme 2 may explain the relatively high susceptibility of LUAD patients to SARS-CoV-2 infection. CTSB was highly expressed in the LUAD tissues after SARS-CoV-2 infection, and its expression was positively correlated with immune cell infiltration and proinflammatory cytokine expression. These findings suggest that CTSB plays a vital role in the hyperinflammatory response in COVID-19 patients with LUAD and is a promising target for the development of a novel drug therapy for COVID-19 patients.

FURIN correlated with immune infiltration serves as a potential biomarker in SARS-CoV-2 infection-related lung adenocarcinoma.

FURIN, as a proprotein convertase, has been found to be expressed in a variety of cancers and plays an important role in cancer. In addition, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires FURIN to enter human cells. However, the role of FURIN in lung adenocarcinoma remains unclear. And the expression of SARS-CoV-2 related gene in lung adenocarcinoma has not been clarified. Therefore, in order to explore the prognostic value and mechanism of FURIN in lung adenocarcinoma, we performed bioinformatics analysis with Oncomine, Tumor Immune Estimation Resource, Gene Expression Profiling Interactive Analysis, human protein atlas, UALCAN, PrognoScan, Kaplan-Meier plotter, cBioPortal and LinkedOmics databases. And then we used GSE44274 in the GEO (Gene Expression Omnibus) database to analyze the expression of FURIN in LUAD patients who infected with SARS-CoV. FURIN was highly expressed in lung adenocarcinoma and was significantly associated with poor overall survival. FURIN expression was found to be correlated with six major permeable immune cells and with macrophage immune marker in LUAD patients. In addition, SARS-CoV-2 infection might affect the expression of FURIN. FURIN can be used as a promising biomarker for determining prognosis and immune infiltration in LUAD patients.

Oncolytic Bovine Herpesvirus 1 Inhibits Human Lung Adenocarcinoma A549 Cell Proliferation and Tumor Growth by Inducing DNA Damage.

Bovine herpesvirus 1 (BoHV-1) is a promising oncolytic virus with broad antitumor spectrum; however, its oncolytic effects on human lung adenocarcinoma in vivo have not been reported. In this study, we report that BoHV-1 can be used as an oncolytic virus for human lung adenocarcinoma, and elucidate the underlying mechanism of how BoHV-1 suppresses tumor cell proliferation and growth. First, we examined the oncolytic activities of BoHV-1 in human lung adenocarcinoma A549 cells. BoHV-1 infection reduced the protein levels of histone deacetylases (HDACs), including HDAC1-4 that are promising anti-tumor drug targets. Furthermore, the HDAC inhibitor Trichostatin A (TSA) promoted BoHV-1 infection and exacerbated DNA damage and cytopathology, suggesting a synergy between BoHV-1 and TSA. In the A549 tumor xenograft mouse model, we, for the first time, showed that BoHV-1 can infect tumor and suppressed tumor growth with a similar high efficacy as the treatment of TSA, and HDACs have potential effects on the virus replication. Taken together, our study demonstrates that BoHV-1 has oncolytic effects against human lung adenocarcinoma in vivo.

Gene signatures and potential therapeutic targets of Middle East respiratory syndrome coronavirus (MERS-CoV)-infected human lung adenocarcinoma epithelial cells.

BACKGROUND: Pathogenic coronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. These viruses have induced outbreaks worldwide, and there are currently no effective medications against them. Therefore, there is an urgent need to develop potential drugs against coronaviruses. METHODS: High-throughput technology is widely used to explore differences in messenger (m)RNA and micro (mi)RNA expression profiles, especially to investigate protein-protein interactions and search for new therapeutic compounds. We integrated miRNA and mRNA expression profiles in MERS-CoV-infected cells and compared them to mock-infected controls from public databases. RESULTS: Through the bioinformatics analysis, there were 251 upregulated genes and eight highly differentiated miRNAs that overlapped in the two datasets. External validation verified that these genes had high expression in MERS-CoV-infected cells, including RC3H1, NF-κB, CD69, TNFAIP3, LEAP-2, DUSP10, CREB5, CXCL2, etc. We revealed that immune, olfactory or sensory system-related, and signal-transduction networks were discovered from upregulated mRNAs in MERS-CoV-infected cells. In total, 115 genes were predicted to be related to miRNAs, with the intersection of upregulated mRNAs and miRNA-targeting prediction genes such as TCF4, NR3C1, and POU2F2. Through the Connectivity Map (CMap) platform, we suggested potential compounds to use against MERS-CoV infection, including diethylcarbamazine, harpagoside, bumetanide, enalapril, and valproic acid. CONCLUSIONS: The present study illustrates the crucial roles of miRNA-mRNA interacting networks in MERS-CoV-infected cells. The genes we identified are potential targets for treating MERS-CoV infection; however, these could possibly be extended to other coronavirus infections.

Torque Teno Virus load in lung cancer patients correlates with age but not with tumor stage.

BACKGROUND: Torque teno virus (TTV) is a ubiquitous non-pathogenic virus, which is suppressed in immunological healthy individuals but replicates in immune compromised patients. Thus, TTV load is a suitable biomarker for monitoring the immunosuppression also in lung transplant recipients. Since little is known about the changes of TTV load in lung cancer patients, we analyzed TTV plasma DNA levels in lung cancer patients and its perioperative changes after lung cancer surgery. MATERIAL AND METHODS: Patients with lung cancer and non-malignant nodules as control group were included prospectively. TTV DNA levels were measured by quantiative PCR using DNA isolated from patients plasma and correlated with routine circulating biomarkers and clinicopathological variables. RESULTS: 47 patients (early stage lung cancer n = 30, stage IV lung cancer n = 10, non-malignant nodules n = 7) were included. TTV DNA levels were not detected in seven patients (15%). There was no significant difference between the stage IV cases and the preoperative TTV plasma DNA levels in patients with early stage lung cancer or non-malignant nodules (p = 0.627). While gender, tumor stage and tumor histology showed no correlation with TTV load patients below 65 years of age had a significantly lower TTV load then older patients (p = 0.022). Regarding routine blood based biomarkers, LDH activity was significantly higher in patients with stage IV lung cancer (p = 0.043), however, TTV load showed no correlation with LDH activity, albumin, hemoglobin, CRP or WBC. Comparing the preoperative, postoperative and discharge day TTV load, no unequivocal pattern in the kinetics were. CONCLUSION: Our study suggest that lung cancer has no stage dependent impact on TTV plasma DNA levels and confirms that elderly patients have a significantly higher TTV load. Furthermore, we found no uniform perioperative changes during early stage lung cancer resection on plasma TTV DNA levels.

HPV and lung cancer: A systematic review and meta-analysis.

BACKGROUND: Lung cancer has emerged as a global public health problem and is the most common cause of cancer deaths by absolute cases globally. Besides tobacco, smoke infectious diseases such as human papillomavirus (HPV) might be involved in the pathogenesis of lung cancer. However, data are inconsistent due to differences in study design and HPV detection methods. AIM: A systematic meta-analysis was performed to examine the presence of HPV-infection with lung cancer. METHODS AND RESULTS: All studies in all languages were considered for the search concepts "lung cancer" and "HPV" if data specific to HPV prevalence in lung cancer tissue were given. This included Journal articles as well as abstracts and conference reports. As detection method, only HPV PCR results from fresh frozen and paraffin-embedded tissue were included. Five bibliographic databases and three registers of clinical trials including MEDLINE, Embase, Cochrane Library, and ClinicalTrials.gov were searched through February 2020. A total 4298 publications were identified, and 78 publications were selected, resulting in 9385 included lung cancer patients. A meta-analysis of 15 case-control studies with n = 2504 patients showed a weighted overall prevalence difference of 22% (95% CI: 12%-33%; P < .001) and a weighted overall 4.7-fold (95% CI: 2.7-8.4; P < .001) increase of HPV prevalence in lung cancer patients compared to controls. Overall, HPV prevalence amounted to 13.5% being highest in Asia (16.6%), followed by America (12.8%), and Europe (7.0%). A higher HPV prevalence was found in squamous cell carcinoma (17.9%) compared to adenocarcinoma (P < .01) with significant differences in geographic patterns. HPV genotypes 16 and 18 were the most prevalent high-risk genotypes identified. CONCLUSION: In conclusion, our review provides convincing evidence that HPV infection increases the risk of developing lung cancer.

Preliminary assessment of viral metagenome from cancer tissue and blood from patients with lung adenocarcinoma.

In this study, using a viral metagenomic method, we investigated the composition of virome in blood and cancer tissue samples that were collected from 25 patients with lung adenocarcinoma. Results indicated that virus sequences showing similarity to human pegivirus (HPgV), anellovirus, human endogenous retrovirus (HERV), and polyomavirus were recovered from this cohort. Three different complete genomes of HPgV were acquired from the blood samples and one complete genome of polyomavirus was determined from the cancer tissue sample. Phylogenetic analysis indicated that the three HPgV strains belonged to genotype 3 and the polyomavirus showed the highest sequence identity (99.73%) to trichodysplasia spinulosa-associated polyomavirus. PCR screening results indicated that the three HPgVs were present in 5 out of the 25 blood samples and the polyomavirus only existed in a cancer tissue sample pool. Whether infections with viruses have an association with lung cancer needs further study with a larger size of sampling.

Lung adenocarcinoma patients have higher risk of SARS-CoV-2 infection.

Both lung adenocarcinoma and coronavirus disease 2019 would cause pulmonary inflammation. Angiotensin-converting enzyme 2, the functional receptor of SARS-CoV-2, also plays a key role in lung adenocarcinoma. To study the risk of SARS-CoV-2 infection in lung adenocarcinoma patients, mRNA and microRNA profiles were obtained from The Cancer Genome Atlas and Gene Expression Omnibus followed by bioinformatics analysis. A network which regards angiotensin-converting enzyme 2 as the center was structured. In addition, via immunological analysis to explore the essential mechanism of SARS-CoV-2 susceptibility in lung adenocarcinoma. Compared with normal tissue, angiotensin-converting enzyme 2 was increased in lung adenocarcinoma patients. Furthermore, a total of 7 correlated differently expressed mRNAs (ACE2, CXCL9, MMP12, IL6, AZU1, FCN3, HYAL1 and IRAK3) and 5 correlated differently expressed microRNAs (miR-125b-5p, miR-9-5p, miR-130b-5p, miR-381-3p and miR-421) were screened. Interestingly, the most frequent toll-like receptor signaling pathway was enriched by mRNA (interlukin 6) and miRNA (miR-125b-5p) sets simultaneously. In conclusion, it was assumed that miR-125b-5p-ACE2-IL6 axis could alter the risk of SARS-CoV-2 infection in lung adenocarcinoma patients.

Expression and Clinical Significance of SARS-CoV-2 Human Targets in Neoplastic and Non-Neoplastic Lung Tissues.

BACKGROUND: A higher incidence of COVID-19 infection was demonstrated in cancer patients, including lung cancer patients. This study was conducted to get insights into the enhanced frequency of COVID-19 infection in cancer. METHODS: Using different bioinformatics tools, the expression and methylation patterns of ACE2 and TMPRSS2 were analyzed in healthy and malignant tissues, focusing on lung adenocarcinoma and data were correlated to clinical parameters and smoking history. RESULTS: ACE2 and TMPRSS2 were heterogeneously expressed across 36 healthy tissues with the highest expression levels in digestive, urinary and reproductive organs, while the overall analysis of 72 paired tissues demonstrated significantly lower expression levels of ACE2 in cancer tissues when compared to normal counterparts. In contrast, ACE2, but not TMPRSS2, was overexpressed in LUAD, which inversely correlated to the promoter methylation. This upregulation of ACE2 was age-dependent in LUAD, but not in normal lung tissues. TMPRSS2 expression in non-neoplastic lung tissues was heterogeneous and dependent on sex and smoking history, while it was downregulated in LUAD of smokers. Cancer progression was associated with a decreased TMPRSS2 but unaltered ACE2. In contrast, ACE2 and TMPRSS2 of lung metastases derived from different cancer subtypes was higher than organ metastases of other sites. TMPRSS2, but not ACE2, was associated with LUAD patients' survival. CONCLUSIONS: Comprehensive molecular analyses revealed a heterogeneous and distinct expression and/or methylation profile of ACE2 and TMPRSS2 in healthy lung vs. LUAD tissues across sex, age and smoking history and might have implications for COVID-19 disease.

Exogenous Jaagsiekte Sheep Retrovirus type 2 (exJSRV2) related to ovine pulmonary adenocarcinoma (OPA) in Romania: prevalence, anatomical forms, pathological description, immunophenotyping and virus identification.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. RESULTS: Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). CONCLUSIONS: In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.

Dyspnea in an HIV Patient: A Not so Typical Presentation of Lung Adenocarcinoma.

Dyspnea in a HIV patient often warrants an extensive workup. The most common etiology of this presentation is likely due to an infectious etiology. However, with the introduction of antiretroviral treatment, non-AIDS-defining illness including malignancies are increasingly being reported. We report the case of a 46-year-old African American female, nonsmoker who presented with dyspnea and found to have pericardial effusion. In patients with HIV presenting with dyspnea, pericardial effusion should be considered among the differential diagnosis, more so in patients in whom infectious etiologies have been ruled out. Further workup, including imaging and biopsy, revealed that our patient had metastatic lung adenocarcinoma. The introduction of antiretroviral treatment has significantly reduced mortality for those with AIDS from AIDS-defining illness and malignancies. However, the incidence of non-AIDS-defining malignancies like lung adenocarcinoma (most common non-AIDS-defining malignancy) is being increasingly reported. Lung adenocarcinoma often presents at a younger age in patients with HIV than the general population. Smoking rates are higher in patients with HIV and may be a contributing factor to the early onset of lung cancer; however, other factors such as long-term medications and immunomodulation in HIV may also play a role. Prognosis is also worse for HIV-positive patients having lung cancer compared with those who are HIV negative, even at a similar stage of cancer.

Human papillomavirus infection maybe not associated with primary lung cancer in the Fujian population of China.

BACKGROUND: To investigate whether human papillomavirus (HPV) infection is associated with primary lung cancer among the Fujian population. METHODS: HPV infection was detected in 140 pairs of lung cancer tissues and matched paracancerous tissues by examining the 21 clinically relevant HPV types using a combination of viral highly conserved L1 region PCR amplification and specific probe reverse hybridization. Paired χ2 test was used to analyze differences in detection rates of HPV between lung cancer and paracancerous tissues. Differences in detection rates of HPV in lung cancer tissues were analyzed using χ2 test or the exact probability method. The rank sum test was used to analyze differences in the distributions of routine indices of blood and pulmonary function in lung cancer tissues between the HPV negative and positive groups. RESULTS: HPV infection was detected in 13 of the 140 tumor specimens and in 16 of the paired normal lung tissues. There was no significant correlation between HPV infection and lung cancer (P > 0.05). The diagnosed HPV infection rates did not differ significantly among lung cancer tissues with different stratification (P > 0.05). However, the platelet count, platelet pressure, residual gas volume, functional residual volume, and residual gas volume/lung total distribution may differ between HPV-negative and HPV-positive lung cancer tissues (0.000625 < P < 0.05). CONCLUSIONS: We concluded that HPV infection may not be associated with the risk of primary lung cancer in the Fujian population. However, HPV infection may affect platelet and residual lung function in primary lung cancer patients.

Expression of p16 and p53 in non-small-cell lung cancer: clinicopathological correlation and potential prognostic impact.

Aim: p16 and p53 are frequently altered intracellular pathways in cancers. We investigated the aberrant expression of p16 and its relationship with p53 and HPV status in primary non-small-cell lung carcinoma. Patients & methods: Lung tumor tissue microarray (n = 163), immunohistochemical study of p16 and p53, and HPV in-situ hybridization were analyzed. Results: p16 and p53 were detected in 50.7 and 57.3% of adenocarcinoma (ADCs; n = 75), and 35.2 and 63.6% of squamous cell carcinoma (n = 88). HPV was detected in 16 and 10.2% of ADC and squamous cell carcinoma. In ADCs, p16 positive tumors demonstrated a favorable median overall survival time of 60.9 months, compared with p16 negative tumors of 46.9 months (p < 0.05). Furthermore, we did not find significant relationships between p16 expression and HPV status, nor with p53 expression. Conclusion: p16 play an unique role in lung cancer survival. The mechanism of p16 needs to be further studied.

Genomic alterations of ground-glass nodular lung adenocarcinoma.

In-depth molecular pathogenesis of ground-glass nodular lung adenocarcinoma has not been well understood. The objectives of this study were to identify genomic alterations in ground-glass nodular lung adenocarcinomas and to investigate whether viral transcripts were detected in these tumors. Nine patients with pure (n = 4) and part-solid (n = 5) ground-glass nodular adenocarcinomas were included. Six were females with a median age of 58 years. We performed targeted exon sequencing and RNA sequencing. EGFR (n = 10), IDH2 (n = 2), TP53 (n = 1), PTEN (n = 1), EPHB4 (n = 1), and BRAF (n = 1) were identified as driver mutations by targeted exon sequencing. Vasculogenesis-associated genes including NOTCH4 and TGFBR3 expression were significantly downregulated in adenocarcinoma tissue versus normal tissue (adjusted P values < 0.001 for both NOTCH4 and TGFBR3). In addition, five novel fusion gene loci were identified in four lung adenocarcinomas. However, no significant virus-associated transcripts were detected in tumors. In conclusions, EGFR, IDH2, TP53, PTEN, EPHB4, and BRAF were identified as putative driver mutations of ground-glass nodular adenocarcinomas. Five novel fusion genes were also identified in four tumors. Viruses do not appear to be involved in the tumorigenesis of ground-glass nodular lung adenocarcinoma.