Human Lung Cancer Patients Research Articles

A Novel Inverse Algorithm To Solve the Integrated Optimization of Dose, Dose Rate, and Linear Energy Transfer of Proton FLASH Therapy With Sparse Filters

Purpose:The recently proposed Integrated Physical Optimization Intensity Modulated Proton Therapy (IPO-IMPT) framework allows simultaneous optimization of dose, dose rate, and linear energy transfer (LET) for FLASH treatment planning. Finding solutions to IPO-IMPT is difficult due to computational intensiveness. Nevertheless, an inverse solution that simultaneously specifies the geometry of a sparse filter and weights of a proton intensity map is desirable for both clinical and preclinical applications. Such solutions can reduce effective biological dose to organs at risk in cancer patients as well as reduce the number of animal irradiations needed to derive extra biological dose models in preclinical studies. Methods:Unlike the initial forward heuristic, this inverse IPO-IMPT solution includes simultaneous optimization of sparse range compensation, sparse range modulation, and spot intensity. The daunting computational tasks vital to this endeavor were resolved iteratively with a distributed computing framework to enable Simultaneous Intensity and Energy Modulation and Compensation (SIEMAC). SIEMAC was demonstrated on a human central lung cancer patient and a minipig. Results:SIEMAC simultaneously improves maps of spot intensities and patient-field-specific sparse range compensators and range modulators. For the lung cancer patient, at our maximum nozzle current of 300 nA, dose rate coverage above 100 Gy/s increased from 57% to 96% in the lung and from 93% to 100% in the heart, and LET coverage above 4 keV/μm dropped from 68% to 9% in the lung and from 26% to <1% in the heart. For a simple minipig plan, the full-width-half-maximum of the dose, dose rate, and LET distributions decreased by 30%, 1.6%, and 57%, respectively, again with similar target dose coverage, thus reducing uncertainty in these quantities for preclinical studies. Conclusion:The inverse solution to IPO-IMPT demonstrated the capability to simultaneously modulate sub-spot proton energy and intensity distributions for clinical and preclinical studies.

The IL-25/ILC2 axis promotes lung cancer with a concomitant accumulation of immune-suppressive cells in tumors in humans and mice.

Group 2 innate lymphoid cells (ILC2) can be activated by interleukin (IL)-33 or IL-25. IL-25-activated ILC2 cells help protect the host against helminth infection while exacerbating allergic-like inflammation and tissue damage in the lung. In the context of cancer, IL-33-activated ILC2 cells were found to bear anti-tumoral functions in lung cancer while IL-25-activated ILC2 cells promoted tumorigenesis in colorectal cancer. The role of IL-25-activated ILC2 cells in lung cancer remains to be addressed. We examined the overall survival of human non-small cell lung cancer (NSCLC) patients according to IL25 expression as well as the distribution of ILC2 cells and regulatory T cells (Tregs) in various NSCLC patient tissues and peripheral blood (PB) of healthy donors (HDs). We analyzed the effect of adoptive transfer of IL-25-activated ILC2 cells on tumor growth, metastasis and survival in a heterotopic murine model of lung cancer. We report that human NSCLC patients with high IL-25 expression have reduced overall survival. Moreover, NSCLC patients bear increased frequencies of ILC2s compared to HDs. Frequencies of Tregs were also increased in NSCLC patients, concomitantly with ILC2s. In mice bearing heterotopic lung cancer, adoptive transfer of IL-25-activated ILC2s led to increased tumor growth, increased metastasis and reduced survival. The frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs) were found to be increased in the tumors of mice that received ILC2s as compared to controls. Overall, our results indicate that the IL-25/ILC2 axis promotes lung cancer potentially by recruiting immune-suppressive cells to the tumors both in humans and in mice, and that it may therefore represent a suitable novel target for NSCLC immunotherapeutic development.

Cigarette smoking induces lung cancer tumorigenesis via upregulation of the WNT/β-catenin signaling pathway

Lung cancer has the highest mortality rate compared to any other cancer worldwide, and cigarette smoking is one of the major etiological factors. How cigarette smoke (CS) induces tumorigenesis in healthy cells is still not completely understood. In this study, we treated healthy human bronchial epithelial cells (16HBE14o) with 1 % cigarette smoke extract (CSE) for one week. The CSE exposed cells showed upregulation of WNT/β-catenin pathway genes like WNT3, DLV3, AXIN and β-catenin, 30 oncology proteins were found to be upregulated after CSE treatment. Further, we explored whether the role of extracellular vesicles (EVs) obtained from CSE exposed cells can induce tumorigenesis. We observed that CSE EVs induced migration of healthy 16HBE14o cells by upregulation of various oncology proteins in recipient cells like AXL, EGFR, DKK1, ENG, FGF2, ICAM1, HMOX1, HIF1a, SERPINE1, SNAIL, HGFR, PLAU which are related to WNT signaling, epithelial mesenchymal transition (EMT) and Inflammation, whereas inflammatory marker, GAL-3 and EMT marker, VIM were downregulated. Moreover, β-catenin RNA was found in CSE EVs, upon treatment of these EVs to healthy cells, the β-catenin gene level was decreased in recipient cells compared to healthy 16HBE14o cells, indicating the utilisation of β-catenin RNA in healthy cells. Overall, our study suggests that CS treatment can induce tumorigenesis of healthy cells by upregulating WNT/β-catenin signaling in vitro and human lung cancer patients. Therefore targeting the WNT/β-catenin signaling pathway is involved in tumorigenesis inhibition of this pathway could be a potential therapeutic approach for cigarette smoke induced lung cancer.

Pathogenesis of Tobacco-Associated Lung Adenocarcinoma Is Closely Coupled with Changes in the Gut and Lung Microbiomes.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a−/− mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a−/− mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

Topoisomerase I poison-triggered immune gene activation is markedly reduced in human small-cell lung cancers by impairment of the cGAS/STING pathway

BackgroundCurrent immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers.MethodsWe used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines.ResultsTOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets.ConclusionsAltogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours.

Arvanil, a synthetic capsaicin mimetic, synergizes with Irinotecan to trigger enhanced apoptosis in Cisplatin‐resistant Human Lung Cancer

Cisplatin‐based combination therapy is the first line treatment for human lung cancer patients. Initially, cisplatin shows excellent therapeutic response inducing remission in about 80% of lung cancer patients; however, the tumor inevitably relapses (within a year) and this relapsed tumor is resistant to cisplatin. Patients with platinum‐resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, irinotecan has an objective response rate of approximately 3% and little or no survival benefit. Therefore, agents which improve the therapeutic response (of human lung cancers) towards irinotecan may be useful for the treatment of cisplatin‐resistant human lung cancer. Our published data show that the nutritional compound capsaicin sensitized human lung cancer cells towards the pro‐apoptotic activity of camptothecin (an analog of irinotecan) in cell culture and athymic mouse models. The administration of capsaicin causes stomach cramps, gastrointestinal irritation, gut pain and vomiting in patients. Arvanil is a non‐pungent synthetic capsaicin‐analog which displays potent growth‐suppressive activity in human lung cancer. The present study aims to investigate the combinatorial apoptotic activity of arvanil and irinotecan in cisplatin‐resistant lung cancer. Caspase‐3 activity assays reveal that the combination of irinotecan and arvanil displayed greater apoptotic activity in H69‐CPR human cisplatin‐resistant SCLC cells than the drugs used alone. These experiments were repeated in a second cisplatin‐resistant lung cancer cell line PC9‐CDDP and similar results were obtained. Chou‐Talalay isobologram analysis showed the interaction between irinotecan and arvanil is synergistic in H69‐CPR and PC9‐CDDP cells. The combination of arvanil/irinotecan showed a greater anti‐tumor activity in H69‐CPR tumors xenografted on chicken chorioallantoic membrane (CAM) and in SCID mice. In summary, the results of our studies pave the way for novel combination therapies for the management and treatment of drug resistant lung cancers in patients.

Autophagy-mediated degradation of NOTCH1 intracellular domain controls the epithelial to mesenchymal transition and cancer metastasis

BackgoundAutophagy controls levels of cellular components during normal and stress conditions; thus, it is a pivotal process for the maintenance of cell homeostasis. In cancer, autophagy protects cells from cancerous transformations that can result from genomic instability induced by reactive oxygen species or other damaged components, but it can also promote cancer survival by providing essential nutrients during the metabolic stress condition of cancer progression. However, the molecular mechanism underlying autophagy-dependent regulation of the epithelial to mesenchymal transition (EMT) and metastasis is still elusive.MethodsThe intracellular level of NOTCH1 intracellular domain (NICD) in several cancer cells was studied under starvation, treatment with chloroquine or ATG7-knockdown. The autophagy activity in these cells was assessed by immunocytochemistry and molecular analyses. Cancer cell migration and invasion under modulation of autophagy were determined by in vitro scratch and Matrigel assays.ResultsIn the study, autophagy activation stimulated degradation of NICD, a key transcriptional regulator of the EMT and cancer metastasis. We also found that NICD binds directly to LC3 and that the NICD/LC3 complex associates with SNAI1 and sequestosome 1 (SQSTM1)/p62 proteins. Furthermore, the ATG7 knockdown significantly inhibited degradation of NICD under starvation independent of SQSTM1-associated proteasomal degradation. In addition, NICD degradation by autophagy associated with the cellular level of SNAI1. Indeed, autophagy inhibited nuclear translocation of NICD protein and consequently decreased the transcriptional activity of its target genes. Autophagy activation substantially suppressed in vitro cancer cell migration and invasion. We also observed that NICD and SNAI1 levels in tissues from human cervical and lung cancer patients correlated inversely with expression of autophagy-related proteins.ConclusionsThese findings suggest that the cellular level of NICD is regulated by autophagy during cancer progression and that targeting autophagy-dependent NICD/SNAI1 degradation could be a strategy for the development of cancer therapeutics.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for invivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

Abstract 2140: Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC

Abstract Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second enzymatic step within the serine synthetic pathway and its expression is elevated in numerous human cancers, such as NSCLC. While PSAT1 suppression decreases SSP activity and cell proliferation, non-canonical activities have recently been suggested that may contribute to its pro-tumorigenic function, as is seen with other metabolic enzymes such as pyruvate kinase. Thus, we conducted a genome wide expression screen to uncover potential unknown cellular processes impacted by PSAT1 and to identify a PSAT1-associated gene signature that might inform patient outcomes in NSCLC. Loss of PSAT1 in EGFR-mutant PC9 lung cancer cells led to 491 differentially expressed genes (279-down; 212-up). KEGG and gene ontology analysis identified biological pathways such as metabolic processes, cell cycle regulation, cell adhesion, and cell motility. Transcript analysis confirmed inhibitory effects on the Rb/E2F1 and β-catenin pathways as well as expression of effectors on the actin cytoskeleton upon loss of PSAT1. Importantly, these could be rescued by PSAT1 restoration, including the loss of F-actin stress fibers as observed with phalloidin staining. Lastly, a bioinformatics approach utilizing transcriptomic data from human lung cancer patients was used to define genes altered between EGFR-mutant lung cancers and normal lung and that were differentially expressed in our PSAT1 depletion RNA-seq study. From this, we identified a PSAT1-associated gene signature that was then used to assign risk survival scores to these NSCLC patients. Subsequent, Kaplan Meier analysis demonstrated the prognostic ability of this gene signature for both relapse free and overall survival in EGFR-mutant NSCLC but was unable to predict risk groups in other NSCLC subtypes. Together, transcriptomic analysis under PSAT1 silencing identified its impact on various biological processes, including actin cytoskeleton rearrangement, and led to identification of a gene signature that has predictive ability towards survival outcomes in EGFR-mutant lung cancer. Citation Format: Rumeysa Biyik-Sit, Brian F. Clem. Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2140.

Antitumor Activities of Immune Costimulatory Molecules of TNF Superfamily in Colorectal and Lung Cancers

Cancer immunotherapy has emerged as a promising treatment that utilizes the innate or adaptive immunity to generate a robust killing of malignant cells. However, only a limited number of cancer patients showed responsiveness to immunotherapies such as checkpoint inhibitors, suggesting the need for potent alternative strategies. In the present study, we explored the therapeutic potentials of costimulatory molecules including TNF superfamily member 4 (TNFSF4), member 9 (TNFSF9), and member 18 (TNFSF18). In tumor samples from human colorectal and lung cancer patients, expression of these factors positively correlated with lymphocyte infiltration and expression of several immune effector genes. In syngeneic mouse tumor models, overexpression of the TNF superfamily costimulatory factors in murine colorectal or lung cancer cells significantly suppressed tumor progression. Especially, TNFSF9 and 18 showed stronger antitumor effects than TNFSF4. Together, our study demonstrated the great potential of cancer immunotherapy targeting these immune costimulatory molecules.

Fibronectin regulates anoikis resistance via cell aggregate formation.

The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

52350 PKM2 mediates anti-tumor immunity and T cell dysfunction

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.

De novo necroptosis creates an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors

Cancer immunotherapies using monoclonal antibodies to block inhibitory checkpoints are showing durable remissions in many types of cancer patients, although the majority of breast cancer patients acquire little benefit. Human melanoma and lung cancer patient studies suggest that immune checkpoint inhibitors are often potent in patients that already have intratumoral T cell infiltrate; although it remains unknown what types of interventions can result in an intratumoral T cell infiltrate in breast cancer. Using non-T cell-inflamed mammary tumors, we assessed what biological processes and downstream inflammation can overcome the barriers to spontaneous T cell priming. Here we show a specific type of combination therapy, consisting of oncolytic virus and chemotherapy, activates necroptosis and limits tumor growth in autochthonous tumors. Combination therapy activates proinflammatory cytokines; intratumoral influx of myeloid cells and cytotoxic T cell infiltrate in locally treated and distant autochthonous tumors to render them susceptible to immune checkpoint inhibitors.

MicroRNA-200b is a potential biomarker of the expression of PD-L1 in patients with lung cancer.

BackgroundAdvanced non‐small cell lung cancer (NSCLC) has a high mortality rate and poor prognosis. However, outcomes have gradually improved after the introduction of novel immunotherapies, including immune checkpoint inhibitors (ICIs). Although programmed death‐ligand 1 (PD‐L1) expression in tumor tissues is a known biomarker for guiding ICI treatment of NSCLC, challenges such as difficulty of liquid biopsy and heterogeneous results during treatment persist. This study evaluated the potential of miR200b as a surrogate biomarker for PD‐L1 expression.MethodsWe used the human lung cancer cell lines H226, H460, H520, A549, and H1975. miR200b expression in blood and bronchoscopy specimens of NSCLC patients was evaluated using reverse‐transcription‐quantitative PCR. Using flow cytometry, PD‐L1 expression in vitro, as well as in tumor tissues, was evaluated after transfection with a mimic miR200b or siRNA.ResultsmiR200b expression negatively correlated with PD‐L1 expression in all cell lines. The induction or knockdown of miR200b also altered PD‐L1 expression in vitro. The patient group with a PD‐L1 tumor proportion score ≥ 50% had significantly lower miR200b expression in the bronchoscopy specimens (P = 0.025) and serum‐derived exosomes (P = 0.022) than that with PD‐L1 tumor proportion score < 50%.ConclusionsmiR200b can regulate PD‐L1 expression in lung cancer cells, and miR200b expression in clinical specimens negatively correlated with PD‐L1 expression. Thus, miR200b may be a useful surrogate biomarker for PD‐L1 expression in lung cancer patients.Key pointsSignificant findings of the study High PD‐L1 expression was linked to low miR200b expression, whereas low PD‐L1 expression was linked to high miR200b expression in human lung cancer patients. Thus, miR200b overexpression or silencing can control PD‐L1 expression in cancer cells. What this study adds We demonstrated the potential of miR200b as a surrogate biomarker for PD‐L1 expression in lung cancer patients.

Ubiquilin proteins regulate EGFR levels and activity in lung adenocarcinoma cells.

Ubiquilin (UBQLN) proteins are involved in diverse cellular processes like endoplasmic reticulum-associated degradation, autophagy, apoptosis, and epithelial-to-mesenchymal transition. UBQLNs interact with a variety of substrates, including cell surface receptors, transcription factor regulators, proteasomal machinery proteins, and transmembrane proteins. In addition, previous work from our lab shows that UBQLN1 interacts with insulin-like growth factor receptor family members (IGF1R, IGF2R, and INSR) and this interaction regulates the activity and proteostasis of IGFR family members. We wondered whether UBQLN proteins could also bind and regulate additional receptor tyrosine kinases. Thus, we investigated a link between UBQLN and the oncogene epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells. Loss of UBQLN1 occurs at high frequency in human lung cancer patient samples and we have shown that the loss of UBQLN1 is capable of altering processes involved in cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition in lung adenocarcinoma cell lines. Here, we present data that loss of UBQLN1 resulted in increased turnover of total EGFR while increasing the relative amount of phosphorylated EGFR in lung adenocarcinoma cells, especially in the presence of its ligand EGF. Furthermore, the loss of UBQLN1 led to a more invasive cell phenotype as manifested by increased proliferation, migration, and speed of movement of these lung adenocarcinoma cells. Taken together, UBQLN1 regulates the expression and stability of EGFR in lung cancer cells.

Tizanidine (Hydrochloride) Inhibits A549 Lung Cancer Cell Proliferation and Motility Through Regulating Nischarin.

PurposeTizanidine hydrochloride (TZN) is a centrally acting α2-adrenergic agonist. In this study, we aimed to explore the role of TZN on human lung cancer and to elucidate its underlying mechanisms.MethodsThe effect of TZN treatment in A549 cell proliferation, migration, invasion and apoptosis was evaluated by CCK8, transwell and flow cytometer assays. The expression of apoptosis-related proteins and the activation of AKT and Wnt3a/β-catenin pathways were detected by Western blot. From the data of DrugBank, TZN could act as an agonist to target Nischarin in humans. We next investigated the function of Nischarin receptor in lung cancer and its role in the anti-tumor activity of TZN.ResultsThe treatment of TZN inhibited the proliferation, migration and invasion of A549 cells, and induced apoptosis. These results were further confirmed by that TZN treatment increased the Bax/Bcl-2 ratio in A549 cells. We also observed that TZN treatment changed the expression and phosphorylation of proteins of AKTand Wnt3a/β-catenin signaling pathway members. By bioinformatics analysis, we found that Nischarin was down-regulated in human lung cancer tissues and patients with high Nischarin expression had a better survival. Moreover, Nischarin functioned as a tumor suppressor in the survival and metastasis of A549 cells through the regulation of AKT and Wnt3a/β-catenin pathways. Knockdown of Nischarin promoted the proliferation, invasion, migration of A549 cells and inhibited the apoptosis, which were reversed by the TZN treatment.ConclusionSummary, our data revealed that treatment of TZN inhibited the growth of lung cancer cell line A549 and may be used as a novel strategy for lung cancer therapy.

PD-1 Expression and Function of T-Cell Subsets in TILs From Human Lung Cancer.

On the basis of the autologous tumor-infiltrating lymphocytes (TILs) or genetically modified TILs for adoptive cell therapy have received more attention. Programmed cell death protein 1 (PD-1) expression on the T cells exert complex response during the tumor immune response. But the composition and function of PD-1T-cell subsets in TILs from human lung cancer still limited. In blood and TILs from human lung cancer patients, we confirmed that PD-1 is expressed in higher levels in CD4T-cell subsets than in CD8T-cell subsets. To further analyze the function of PD-1T cells in TILs, we observed the cytokine production in different T-cell subsets. We found that higher interferon-γ and granzyme B production in CD4/CD8PD-1T-cell subsets in TILs than in peripheral blood mononuclear cells (PBMCs); except for PD-1Tscm, higher tumor necrosis factor-α production was observed in PD-1T-cell subsets in TILs than in PBMCs; the expression level of interleukin-17 were lower in PD-1T cells in TILs than in PBMCs; and perforin expression was significantly reduced in CD4PD-1T cells subsets in TILs compared with peripheral blood. Clarify elucidating the composition and function of PD-1T-cell subsets in TILs will have great value in clinical application for evaluating the sensitivity to PD-1 blockade and selecting the promising candidate T-cell subsets in TILs for combination immunotherapy in human lung cancer.

Abstract 5159: Isoform specific expression of cadherin10 is associated with progression and drug resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer related death worldwide accounting for 1.69 million of total 8.8 million cancer deaths in 2015 alone. Nearly 85% of all lung cancer cases are classified as non-small cell lung cancer (NSCLC) with a 5-year survival rate of 16%, despite of all advanced targeted treatment modalities. Like any other cancer, cadherin proteins play a critical role in lung cancer progression, metastasis and drug resistance. Classical type I and type II cadherin proteins are calcium-dependent transmembrane glycoproteins with a cytoplasmic tail that mediates downstream signaling and a large extracellular N-terminal head that engages in homophilic and heterophilic adhesion. While alterations of type I cadherins (N-cadherin and E-cadherin) are well studied, functions as well as alterations of type II cadherins, such as cadherin-8, -9, -10, -12, and -18, remain largely unknown. Recent whole genome sequencing of patient samples towards Precision Medicine initiatives revealed 2,687 donors affected by 9,151 cadherin10 (CDH10) mutations alone across the 63 cancer projects worldwide. The majority of mutations in CDH10 are missense substitutions largely distributed throughout the entire gene length, without any specific hotspot. CDH10 copy number amplification is also reported in a significantly large number of lung cancer patients. Alternative splicing of this gene potentially results in multiple transcript variants. To resolve the conundrum of Loss-of-heterozygosity (LOH) and copy number amplification, we decided to look further in to expression and function of CDH10 using array of lung cancer cell lines and human lung cancer patient samples. Here we report, for the first time, that full-length CDH10 expression as well as the tumor suppressive function are compromised in the course of NSCLC development, while aberrant smaller isoforms appear in the cell lines and tumors that drive the oncogenic progression. Expression of such smaller isoforms were further altered when the cells were treated with commonly used chemotherapy drugs, such as Cisplatin, Doxorubicin and Paclitaxel indicating a direct relationship between drug resistance and appearance of these smaller isoforms. We found that the downregulation of full-length CDH10 using siRNA results in an increase in proliferation and invasion, indicating a potential anti-oncogenic function of full-length CDH10 protein. Therefore, we found that mutations and alternative splicing potentially downregulates full-length CDH10 expression along with its tumor suppressive function. However, appearance of smaller aberrant isoforms accounts for tumorigenic progression and drug resistance. Citation Format: Rajib Ghosh, Phattrakorn Powan, Liying W. Rojanasakul, Yon Rojanasakul. Isoform specific expression of cadherin10 is associated with progression and drug resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5159.

A Novel Translational Ovine Pulmonary Adenocarcinoma Model for Human Lung Cancer.

In vitro cell line and in vivo murine models have historically dominated pre-clinical cancer research. These models can be expensive and time consuming and lead to only a small percentage of anti-cancer drugs gaining a license for human use. Large animal models that reflect human disease have high translational value; these can be used to overcome current pre-clinical research limitations through the integration of drug development techniques with surgical procedures and anesthetic protocols, along with emerging fields such as implantable medical devices. Ovine pulmonary adenocarcinoma (OPA) is a naturally-occurring lung cancer that is caused by the jaagsiekte sheep retrovirus. The disease has similar histological classification and oncogenic pathway activation to that of human lung adenocarcinomas making it a valuable model for studying human lung cancer. Developing OPA models to include techniques used in the treatment of human lung cancer would enhance its translational potential, making it an excellent research tool in assessing cancer therapeutics. In this study we developed a novel OPA model to validate the ability of miniaturized implantable O2 and pH sensors to monitor the tumor microenvironment. Naturally-occurring pre-clinical OPA cases were obtained through an on-farm ultrasound screening programme. Sensors were implanted into OPA tumors of anesthetized sheep using a CT-guided trans-thoracic percutaneous implantation procedure. This study reports the findings from 9 sheep that received sensor implantations. Time taken from initial CT scans to the placement of a single sensor into an OPA tumor was 45 ± 5 min, with all implantations resulting in the successful delivery of sensors into tumors. Immediate post-implantation mild pneumothoraces occurred in 4 sheep, which was successfully managed in all cases. This is, to the best of our knowledge, the first description of the use of naturally-occurring OPA cases as a pre-clinical surgical model. Through the integration of techniques used in the treatment of human lung cancer patients, including ultrasound, general anesthesia, CT and surgery into the OPA model, we have demonstrated its translational potential. Although our research was tailored specifically for the implantation of sensors into lung tumors, we believe the model could also be developed for other pre-clinical applications.

P27Kip1 -p(RhoB)lematic in lung cancer.

Lung cancer is the leading cause of cancer mortality worldwide, with adenocarcinomas of the non‐small cell lung carcinoma (NSCLC) subtype accounting for the majority of cases. Therefore, an urgent need exists for a more detailed dissection of the molecular events driving NSCLC development and the identification of clinically relevant biomarkers. Even though originally identified as a tumour suppressor, recent studies associate the cytoplasmically (mis)localised CDK inhibitor p27Kip1 (p27) with unfavourable responses to chemotherapy and poor outcomes in NSCLC, supporting the hypothesis that the protein can execute oncogenic activities. In a recent issue of The Journal of Pathology, Calvayrac and coworkers uncover a novel molecular mechanism that can explain this oncogenic role of p27. They demonstrate that cytoplasmic p27 binds and inhibits the small GTPase RhoB and thereby relieves a selection pressure for RhoB loss that is frequently observed in NSCLC. This is supported not only by studies with genetically modified mice, but also through identification of a cohort of human lung cancer patients with cytoplasmic p27 and continued RhoB expression, where this signature correlates with decreased survival. This not only establishes a potentially useful biomarker, but also provides yet another facet of the complex roles p27 undertakes in tumourigenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.