Dipeptidyl peptidase 4-positive cancer-associated fibroblasts enhance lung adenocarcinoma growth.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of fibroblasts with various features in the cancer stroma and have been reported to influence cancer progression through cell-cell interactions in various types of malignancies, including lung adenocarcinoma (LUAD). Dipeptidyl peptidase 4 (DPP4) is a transmembrane protein with serine protease activity and is involved in the progression of tumors, metabolic diseases, and autoimmune diseases. In the present study, we focused on the role of DPP4-positive CAFs in LUAD. Immunohistochemistry revealed that 38 of 89 LUAD patients showed DPP4 expression in the fibrous stroma, and patients harboring DPP4-positive CAFs were more often male, had a higher Brinkman index, and had a higher Ki-67 labeling index of tumor cells than those with DPP4-negative CAFs. DPP4-positivity was associated with the expression of other CAF markers, α-SMA, periostin, and podoplanin, as well as a cellular senescence marker, p16. In the in vitro study, conditioned media collected from pulmonary fibroblast (OUS-11, HPF, and HPF-C)-induced overexpression of DPP4 significantly promoted the proliferation of LUAD cells (A549 and PC-9) and increased the expression levels of MCP-1, IL-8, IL-6, and GCSF in the media compared to those in controls. In addition, OUS-11 overexpression in DPP4 overexpression increased periostin expression. In conclusion, DPP4-positive CAFs could promote lung adenocarcinoma cell growth by producing soluble factors, and DPP4 inhibition may inhibit cancer progression.

UBE2V2 promotes metastasis by regulating EMT and predicts a poor prognosis in lung adenocarcinoma.

PURPOSE: As a member of the ubiquitin-conjugating enzyme (E2) family, UBE2V2 demonstrates significant tumorigenicity in many cancers. However, the relationship between UBE2V2 expression and the morbidity of lung adenocarcinoma (LUAD) is still unknown. METHODS: We detected the mRNA and protein expression of UBE2V2 and analyzed its relationship with clinical parameters as well as survival prognosis based on bioinformatic and immunohistochemistry (IHC) in LUAD. The signaling pathway of UBE2V2 in the development of LUAD was obtained by GSEA. The TIMER database was used to investigate the association between UBE2V2 expression and the level of infiltration of different immune cells. Finally, we explored the effects of UBE2V2 knockdown on the proliferation, apoptosis, and migration of LUAD cells. RESULTS: The results showed that UBE2V2 was a potential oncogene and might be considered an independent prognostic molecule for LUAD patients based on TCGA prediction (HR: 1.497 p = 0.012) and IHC (HR:1.864 p = 0.044). IHC showed that UBE2V2 was related to the following clinicopathological factors: gender (p = 0.043), stage (p = 0.042), and lymph node metastasis (p = 0.002). Finally, knockdown of UBE2V2 reduced the migration of LUAD cells by regulating EMT-related proteins. Knockdown of UBE2V2 induced LUAD cells to arrest in the G1 phase. Knockdown of UBE2V2 increased LUAD cell apoptosis and decreased proliferation, which might be related to the downregulation of PCNA and upregulation of P53 and ƳH2AX expression. Interestingly, UBE2V2 is negatively correlated with B cells, CD4+ T cells, macrophages, and dendritic cells. CONCLUSION: UBE2V2 may be a valuable therapeutic target for lung cancer.

Histone deacetylase inhibitors inhibit lung adenocarcinoma metastasis via HDAC2/YY1 mediated downregulation of Cdh1.

Metastasis is a leading cause of mortality in patients with lung adenocarcinoma. Histone deacetylases have emerged as promising targets for anti-tumor drugs, with histone deacetylase inhibitors (HDACi) being an active area of research. However, the precise mechanisms by which HDACi inhibits lung cancer metastasis remain incompletely understood. In this study, we employed a range of techniques, including qPCR, immunoblotting, co-immunoprecipitation, chromatin-immunoprecipitation, and cell migration assays, in conjunction with online database analysis, to investigate the role of HDACi and HDAC2/YY1 in the process of lung adenocarcinoma migration. The present study has demonstrated that both trichostatin A (TSA) and sodium butyrate (NaBu) significantly inhibit the invasion and migration of lung cancer cells via Histone deacetylase 2 (HDAC2). Overexpression of HDAC2 promotes lung cancer cell migration, whereas shHDAC2 effectively inhibits it. Further investigation revealed that HDAC2 interacts with YY1 and deacetylates Lysine 27 and Lysine9 of Histone 3, thereby inhibiting Cdh1 transcriptional activity and promoting cell migration. These findings have shed light on a novel functional mechanism of HDAC2/YY1 in lung adenocarcinoma cell migration.

Histone H3K9 methyltransferase SETDB1 augments invadopodia formation to promote tumor metastasis.

Non-small cell lung cancer (NSCLC) is one of leading causes of cancer-related mortality worldwide, which harbors various accumulated genetic and epigenetic abnormalities. Histone methyltransferase SETDB1 is a pivotal epigenetic regulator whose focal amplification and upregulation are commonly detected in NSCLC. However, molecular mechanisms underlying the pro-oncogenic function of SETDB1 remain poorly characterized. Here, we demonstrate that SETDB1 augments the migration and invasion capabilities of NSCLC cells by reinforcing invadopodia formation and mediated ECM degradation. At the molecular level, SETDB1 suppresses the expression of FOXA2, a crucial tumor and metastasis suppressor via coordinated epigenetic mechanisms - SETDB1 not only catalyzes histone H3K9 methylation on FOXA2 genomic locus, but also recruits DNMT3A to regulate DNA methylation on CpG island. Consequently, depletion of Setdb1 in murine lung adenocarcinoma cells completely abolished their full and spontaneous metastatic capabilities in mouse xenograft models. These findings together establish the pro-metastasis activity of SETDB1 in NSCLC and elucidate the underlying cellular and molecular mechanisms.

Rapid idiosyncratic mechanisms of clinical resistance to KRAS G12C inhibition.

BACKGROUNDThe KRAS proto-oncogene is among the most frequently mutated genes in cancer, yet for 40 years it remained an elusive therapeutic target. Recently, allosteric inhibitors that covalently bind to KRAS G12C mutations have been approved for use in lung adenocarcinomas. Although responses are observed, they are often short-lived, thus making in-depth characterization of the mechanisms of resistance of paramount importance.METHODSHere, we present a rapid-autopsy case of a patient who had a KRASG12C-mutant lung adenocarcinoma who initially responded to a KRAS G12C inhibitor but then rapidly developed resistance. Using deep-RNA and whole-exome sequencing comparing pretreatment, posttreatment, and matched normal tissues, we uncover numerous mechanisms of resistance to direct KRAS inhibition.RESULTSIn addition to decreased KRAS G12C-mutant allele frequency in refractory tumors, we also found reactivation of the MAPK pathway despite no new mutations in KRAS or its downstream mediators. Tumor cell-intrinsic and non-cell autonomous mechanisms included increased complement activation, coagulation, and tumor angiogenesis, and several lines of evidence of immunologic evasion.CONCLUSIONTogether, our findings reveal numerous mechanisms of resistance to current KRAS G12C inhibitors through enrichment of clonal populations, KRAS-independent downstream signaling, and diverse remodeling of the tumor microenvironment.FUNDINGRichard and Fran Duley, Jimmy and Kay Mann, the NIH, and the North Carolina Biotechnology Center.

FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma.

Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.

A MET-PTPRK kinase-phosphatase rheostat controls ZNRF3 and Wnt signaling.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously, we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signaling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/ß-catenin signaling. Conversely, depletion or pharmacological inhibition of MET promotes the internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signaling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signaling that may offer novel opportunities for therapeutic intervention.

Concomitant occurence of multiple autoantibodies against human cytochromes P450.

Cytochromes P450 (CYPs) are a large superfamily of heme-containing enzymes that are essential for the metabolism of a variety of endogenous and xenobiotic compounds. The role and the possible diagnostic or prognostic value of the occurrence of anti-CYP autoantibodies (aAbs) in cancer patients are essentially unclear. Recently we reported the monitoring of aAbs against CYP4Z1 and CYP19A1 in breast cancer patients and healthy controls. In the present study, we extended this investigation by screening the sera of 47 lung cancer patients (17 female and 30 male; age range 49-84) and 119 healthy controls (60 female and 59 male; age range 21-72) for the presence of aAbs directed against CYP2D6, CYP4Z1, or CYP17A1, respectively. Determination of anti-CYP aAb levels was done using our previously established ELISA method. Most sera gave low signals while a small fraction showed stronger responses; however, there were no statistically significant differences between the different test groups. Also, there was no significant difference in aAb signals between the various subtypes of lung cancer. Unexpectedly, sera from two female lung cancer patients (age 67 (adenocarcinoma) and 70 (small cell carcinoma)) and from four healthy controls (one female and three male; age range 34-48) showed significantly elevated signals for more than one of the three CYPs tested. These findings corroborate earlier reports that anti-CYP aAbs occur with low frequency in the general population and, moreover, suggest that the simultaneous presence of multiple aAbs targeting different CYPs should be taken into consideration when evaluating anti-CYP aAbs as biomarkers.

Prostasin regulates PD-L1 expression in human lung cancer cells.

The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of the present study was to investigate if prostasin regulates PD-L1 expression. We established sublines overexpressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-γ (IFNγ) is further enhanced in cells overexpressing the wildtype membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.

Knockdown CYP2S1 inhibits lung cancer cells proliferation and migration.

BACKGROUND: The incidence of lung cancer in Yunnan area ranks firstly in the world and underlying molecular mechanisms of lung cancer in Yunnan region are still unclear. We screened a novel potential oncogene CYP2S1 used mRNA microassay and bioinformation database. The function of CYP2S1 in lung cancer has not been reported. OBJECTIVE: To investigate the functions of CYP2S1 in lung cancer. METHODS: Immunohistochemistry and Real-time PCR were used to verify the expression of CYP2S1. Colony formation and Transwell assays were used to determine cell proliferation, invasion and migration. Xenograft assays were used to detected cell growth in vivo. RESULTS: CYP2S1 is significantly up-regulated in lung cancer tissues and cells. Knockdown CYP2S1 in lung cancer cells resulted in decrease cell proliferation, invasion and migration in vitro. Animal experiments showed downregulation of CYP2S1 inhibited lung cancer cell growth in vivo. GSEA analysis suggested that CYP2S1 played functions by regulating E2F targets and G2M checkpoint pathway which involved in cell cycle. Kaplan-Meier analysis indicated that patients with high CYP2S1 had markedly shorter event overall survival (OS) time. CONCLUSIONS: Our data demonstrate that CYP2S1 exerts tumor suppressor function in lung cancer. The high expression of CYP2S1 is an unfavorable prognostic marker for patient survival.

The intrinsic role and mechanism of tumor expressed-CD38 on lung adenocarcinoma progression.

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8+ T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca2+ and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.

Applicability of pan-TRK immunohistochemistry for identification of NTRK fusions in lung carcinoma.

In the last two decades, various therapies have been introduced for lung carcinoma patients, including tyrosine-kinase inhibitors for different mutations. While some of them are specific to specific tumor types, others, like NTRK1-3 fusions, are found in various solid tumors. The occurrence of an NTRK1,2 or 3 fusion acts as a biomarker for efficient treatment with NTRK inhibitors, irrespectively of the tumor type. However, the occurrence of the NTRK1-3 fusions in lung carcinomas is extremely rare. We performed a retrospective analysis to evaluate the applicability of immunohistochemistry with the pan-TRK antibody in the detection of NTRK fusions in lung carcinomas. The study cohort included 176 adenocarcinomas (AC), 161 squamous cell carcinomas (SCC), 31 large-cell neuroendocrine carcinomas (LCNEC), and 19 small cell lung carcinomas (SCLC). Immunohistochemistry (IHC) was performed using the pan-TRK antibody (clone EPR17341, Ventana) on tissue microarrays, while confirmation for all positive cases was done using RNA-based Archer FusionPlex MUG Lung Panel. On IHC staining, 12/387 samples (3.1%) demonstrated a positive reaction. Ten SCC cases (10/161, 6.2%), and two LCNEC cases (2/31, 6.5%) were positive. Positive cases demonstrated heterogeneous staining of tumor cells, mostly membranous with some cytoplasmic and in one case nuclear pattern. RNA-based sequencing did not demonstrate any NTRK1-3 fusion in our patients' collective. Our study demonstrates that pan-TRK expression in lung carcinoma is very low across different histologic types. NTRK1-3 fusions using an RNA-based sequencing approached could not be detected. This stresses the importance of confirmation of immunohistochemistry results by molecular methods.

E3 ubiquitin ligase PJA1 regulates lung adenocarcinoma apoptosis and invasion through promoting FOXR2 degradation.

Among all lung cancer cases, lung adenocarcinoma (LAC) represents nearly 40% and remains the leading cause of cancer deaths worldwide. Although the combination therapy of surgical treatment with radiotherapy, chemotherapy, and immunotherapy, has been used to treat LAC, unfortunately, high recurrence rates and poor survival remain. Therefore, novel prognostic markers and new targets for molecular targeted therapy in LAC is urgently needed. Fork-head box R2 (FOXR2) plays a key role in a wide range of cellular processes, including cellular proliferation, invasion, differentiation, and apoptosis, and it has been reported to be implicated in progression of LAC, thus inhibition of FOXR2 may be a novel targeting therapy for lung cancer. This current study found that E3 ligase PJA1 regulates ubiquitin-mediated degradation of FOXR2 and predicts good outcome of patients with LAC. In addition, it was showed force expression of PJA1 significantly inhibited LAC cells invasion and induced apoptosis in vitro through inactivating Wnt/ß-catenin signaling pathway. In short, our findings reveal that PJA1 could be a potential diagnostic and prognostic biomarkers and the PJA1- FOXR2 axis could be served as a promising target for LAC therapy.

An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.

When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue. As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell. How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment. Many lung cancers have an identity similar to normal lung cells. However, some turn off a gene that codes for a protein called NKX2-1, which leads to a type of cancer called invasive mucinous adenocarcinoma (or IMA for short). Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach. But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1, and how transitioning to a different cell type impacts their response to treatment. To investigate this, Zewdu et al. studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer. This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors. Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer. These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell. This identity change was found to depend on two signaling pathways which cells use to communicate. This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors. It also shows new relationships between key genes in these cancers and systems for cell communication. These findings could lead to better therapies for lung cancer, particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment.

Potential role of glucosamine-phosphate N-acetyltransferase 1 in the development of lung adenocarcinoma.

Glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is a key enzyme associated with glucose metabolism and uridine diphosphate-N-acetylglucosamine biosynthesis. Abnormal GNPNAT1 expression might be associated with carcinogenesis. We analyzed multiple lung adenocarcinoma (LUAD) gene expression databases and verified GNPNAT1 higher expression in LUAD tumor tissues than in normal tissues. Moreover, we analyzed the survival relationship between LUAD patients' clinical status and GNPNAT1 expression, and found higher GNPNAT1 expression in LUAD patients with unfavorable prognosis. We built GNPNAT1 gene co-expression networks and further annotated the co-expressed genes' Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and various associated regulatory factors. These co-expression genes' functional networks mainly participate in chromosome segregation, RNA metabolic process, and RNA transport. We analyzed GNPNAT1 genetic alterations and co-occurrence networks, and the functional networks of these genes showed that GNPNAT1 participates in multiple steps of cell cycle transition and in the development of some cancers. We assessed the correlation between GNPNAT1 expression and cancer immune infiltrates and showed that GNPNAT1 expression is correlated with several immune cells, chemokines, and immunomodulators in LUAD. We found that GNPNAT1 correlates with LUAD development and prognosis, laying a foundation for further research, especially in immunotherapy.

Immunocytochemical Detection of ALK and ROS1 Rearrangements in Lung Cancer Cytological Samples.

The detection of molecular alterations such as ROS1 and ALK rearrangements is performed as part of the diagnosis of advanced-stage lung adenocarcinoma. These alterations allow the treatments with tyrosine kinase inhibitors. Cytological samples are very useful as up to 40% patients are diagnosed with this type of sample. Here we describe the immunocytochemistry technique usable to reveal the overexpression of ALK or ROS1 tyrosine kinase receptors secondary to ALK and ROS1 rearrangements, respectively.

A novel protein ubiquitination-related five-gene signature predicts overall survival in patients with lung adenocarcinoma.

Protein ubiquitination has been reported to be involved in many biological processes that affect cancer cell growth or death. In this study, we identified differentially expressed E3s/DUB-related genes associated with the prognosis of lung adenocarcinoma and then constructed an E3s/DUB enzyme signature prediction model for the training group and validated its accuracy for prognosis prediction in the validation group. According to our constructed model, all patients were divided into the high- or low-risk group, and a comparison of the two groups revealed that the high-risk group had poorer survival and higher mortality than the low-risk group. The calculated risk score was also an independent prognostic factor when analyzed together with other clinical factors. To explore the functions of the signature genes, we predicted the substrate proteins with which they interact and then performed enrichment analysis. Interestingly, we found that the signature genes were enriched in multiple treatment resistance and immune-related pathways. Therefore, we continued to analyze immune infiltration in the samples and found a variety of differences in immune cell infiltration. According to our constructed model, these differences in immune cell infiltration may predict different immune statuses after grouping and are associated with worse prognosis in high-risk patients.

Vorinostat combined with brigatinib overcomes acquired resistance in EGFR-C797S-mutated lung cancer.

The development of a new generation of tyrosine kinase inhibitors (TKIs) has improved the treatment response in lung adenocarcinomas. However, acquired resistance often occurs due to new epidermal growth factor receptor (EGFR) mutations. In particular, the C797S mutation confers drug resistance to T790M-targeting EGFR TKIs. To address C797S resistance, a promising therapeutic avenue is combination therapy that targets both total EGFR and acquired mutations to increase drug efficacy. We showed that combining vorinostat, a histone deacetylase inhibitor (HDACi), with brigatinib, a TKI, enhanced antitumor effects in primary culture and cell lines of lung adenocarcinomas harboring EGFR L858R/T790M/C797S mutations (EGFR-3M). While EGFR phosphorylation was decreased by brigatinib, vorinostat reduced total EGFR-3M (L858R/T790M/C797S) proteins through STUB1-mediated ubiquitination and degradation. STUB1 preferably ubiquitinated other EGFR mutants and facilitated protein turnover compared to EGFR-WT. The association between EGFR and STUB1 required the functional chaperone-binding domain of STUB1 and was further enhanced by vorinostat. Finally, STUB1 levels modulated EGFR downstream functions. Low STUB1 expression was associated with significantly poorer overall survival than high STUB1 expression in patients harboring mutant EGFR. Vorinostat combined with brigatinib significantly improved EGFR-TKI sensitivity to EGFR C797S by inducing EGFR-dependent cell death and may be a promising therapy in treating C797S-resistant lung adenocarcinomas.

Using Disposable Membrane Cell Collector to Enrich Lung Adenocarcinoma Cells in Bloody Pleural Effusion for Anaplastic Lymphoma Kinase Fusion Gene Detection.

PURPOSE: For anaplastic lymphoma kinase (ALK) gene detection, the centrifugal sedimentation method (CSM) and cell block method (CBM) are commonly used to process samples of bloody pleural effusions (BPEs). However, in practice, the impurity content in the processed samples often affects the results and even leads to the detection failure. The purpose of this study was to establish a cell enrichment method (CEM) by using a disposable membrane cell collector to remove blood and inflammatory cells and enrich lung adenocarcinoma cells in BPE for more efficient RNA extraction and ALK gene detection. MATERIALS AND METHODS: CEM proposed in this study and the traditional CSM and CBM were used to treat BPE samples collected from 37 lung adenocarcinoma patients. A DeNovix DS-11 ultraviolet spectrophotometer was used to measure the concentration and purity of extracted RNA. Amplification refractory mutation systems (ARMS) and ABI 7500 fluorescence qPCR were used to detect ALK gene. Through statistical analysis, the CEM was compared with the CSM and CBM in RNA concentration, purity, and ALK gene detection results. RESULTS: The concentration of RNA extracted by using the CEM was significantly higher than that extracted by using the CBM and CSM (p < 0.001). The purity of RNA extracted by using the CEM was significantly higher than that by the other 2 methods (p = 0.011, p = 0.005). ALK gene testing with PCR was successful in all the samples using the CEM, but 2 cases by the CSM and 1 case by the CBM failed. CONCLUSIONS: Using the disposable membrane cell collector to process BPE of lung adenocarcinoma patients for RNA extraction and ALK gene detection is more effective and successful compared with the traditional methods, and it is suggested to be further applied and popularized in clinical practice.

Akt kinase LANCL2 functions as a key driver in EGFR-mutant lung adenocarcinoma tumorigenesis.

Epidermal growth factor receptor (EGFR) is a key oncogene in lung adenocarcinoma (LUAD). Resistance to EGFR tyrosine kinase inhibitors is a major obstacle for EGFR-mutant LUAD patients. Our gene chip array, quantitative polymerase chain reaction validation, and shRNA-based high-content screening identified the Akt kinase lanthionine synthetase C-like protein 2 (LANCL2) as a pro-proliferative gene in the EGFR-mutant LUAD cell line PC9. Therefore, we investigated whether LANCL2 plays a role in promoting cell proliferation and drug resistance in EGFR-mutant LUAD. In silico clinical correlation analysis using the Cancer Genome Atlas Lung Adenocarcinoma dataset revealed a positive correlation between LANCL2 and EGFR expression and an inverse relationship between LANCL2 gain-of-function and survival in LUAD patients. The EGFR-mutant LUAD cell lines PC9 and HCC827 displayed higher LANCL2 expression than the non-EGFR-mutant cell line A549. In addition, LANCL2 was downregulated following gefitinib+pemetrexed combination therapy in PC9 cells. LANCL2 knockdown reduced proliferation and enhanced apoptosis in PC9, HCC827, and A549 cells in vitro and suppressed murine PC9 xenograft tumor growth in vivo. Notably, LANCL2 overexpression rescued these effects and promoted gefitinib + pemetrexed resistance in PC9 and HCC827 cells. Pathway analysis and co-immunoprecipitation followed by mass spectrometry of differentially-expressed genes in LANCL2 knockdown cells revealed enrichment of several cancer signaling pathways. In addition, Filamin A and glutathione S-transferase Mu 3 were identified as two novel protein interactors of LANCL2. In conclusion, LANCL2 promotes tumorigenic proliferation, suppresses apoptosis, and promotes gefitinib+pemetrexed resistance in EGFR-mutant LUAD cells. Based on the positive association between LANCL2, EGFR, and downstream Akt signaling, LANCL2 may be a promising new therapeutic target for EGFR-mutant LUAD.