Lung Cancer Cell Survival Research Articles

Abstract 1442: Identifying the molecular basis by which βIII-tubulin promotes tumor cell survival and drug resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide with the most common sub-type, non-small cell lung carcinoma (NSCLC), accounting for >80% of cases. Despite advances in treatments, 5-year survival rates for this disease remain dismal. Advanced NSCLC responds poorly to current therapy and the mechanisms underlying its resistance and aggressive nature are not well defined. Microtubules form a component of the cell cytoskeleton and alterations in the expression of microtubule proteins are increasingly correlated with more aggressive and treatment-refractory cancers. βIII-tubulin is a neuronal-specific isotype that is aberrantly expressed in a range of cancers including NSCLC, and has been found to be correlated with reduced patient survival. Our group has recently demonstrated that βIII-tubulin contributes to activation of the PI3K/AKT cell-survival pathway in NSCLC cells[1]. One of the key negative regulators of this pathway is the tumor suppressor PTEN. PTEN can inhibit cell growth, migration and invasion, and a loss of PTEN function is often associated with cancer progression through AKT hyperactivation. Here, using forced-genetics cell models, we show that AKT activation is reduced by βIII-tubulin knockdown, and that re-expression of βIII-tubulin restores these levels in NSCLC cells. Interestingly, we also found that βIII-tubulin knockdown resulted in increased PTEN mRNA along with altered levels of Snail-1/β-catenin, suggesting a possible role for βIII-tubulin in regulating the transcriptional pathway of the PTEN tumor suppressor. Building on these findings, we also identified, through a high-throughput cell-based drug screen, several receptor tyrosine kinase inhibitors that synergize with βIII-tubulin knockdown to reduce NSCLC cell proliferation and tumoroid growth. Together, our data demonstrate an important role for βIII-tubulin in modulating the activation and/or expression levels of signaling proteins which form part of a major pro-survival pathway in NSCLC. In addition, our results suggest that inhibition of βIII-tubulin in combination with drugs that target multiple kinases may offer a novel therapeutic strategy for the treatment of NSCLC to improve patient outcomes. [1] McCarroll et al., TUBB3/betaIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer. Cancer Research. 2015;75:415-425. Citation Format: Simon Brayford, Wee Siang Teo, Amelia Parker, Laura McVeigh, Joshua A. McCarroll, Maria Kavallaris. Identifying the molecular basis by which βIII-tubulin promotes tumor cell survival and drug resistance in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1442.

GLI1 activation is a key mechanism of erlotinib resistance in human non-small cell lung cancer.

Lung cancer is the leading cause of cancer-associated death worldwide. In recent years, the advancement of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapies has provided clinical benefits for lung cancer patients with EGFR mutations. The response to EGFR-TKI varies in patients with lung cancer, and resistance typically develops during the course of the treatment. Therefore, understanding biomarkers which can predict resistance to EGFR-TKI is important. Overexpression of GLI causes activation of the Hedgehog (Hh) signaling pathway and plays a critical role in oncogenesis in numerous types of cancer. In the present study, the role of GLI1 in erlotinib resistance was investigated. GLI1 mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and immunohistochemistry (IHC) in lung cancer cell lines and tumor specimens, respectively. GLI1 mRNA expression levels were found to be positively correlated with the IC50 of erlotinib in 15 non-small cell lung cancer (NSCLC) cell lines. The downregulation of GLI1 using siRNA sensitized lung cancer cells to the erlotinib treatment, whereas the overexpression of GLI1 increased the survival of lung cancer cells in the presence of erlotinib, indicating that Hh/GLI activation may play a critical role in the development of TKI resistance in lung cancer. Combined treatment with erlotinib and a GLI1 inhibitor reduced the cell viability synergistically. A retrospective study of patients with NSCLC treated with erlotinib revealed that those with a high IHC score for GLI1 protein expression had a poorer prognosis. These results indicated that GLI1 is a key regulator for TKI sensitivity, and patients with lung cancer may benefit from the combined treatment of TKI and GLI1 inhibitor.

Inactivation of AKT/NF‑κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor-κB (NF-κB) signaling has often been associated with the induction of non-small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF-κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF-κB activity in TNF-α-activated 293 cells stably transfected with an NF-κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF-κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu-1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro-apoptotic caspase-3 and poly (ADP-ribose) polymerase, as well as the reduction of anti-apoptotic proteins Bcl-xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF-κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase-3, but decreased Bcl-xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated-AKT and phosphorylated-NF-κB, whereas co-treatment with ECL inhibited the cisplatin-induced phosphorylation of AKT and NF-κB, leading to an increased sensitization effect on cisplatin-induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF-κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

MiR-708-5p targets oncogenic prostaglandin E2 production to suppress a pro-tumorigenic phenotype in lung cancer cells.

Many cancers maintain an inflammatory microenvironment to promote their growth. Lung cancer is of particular importance, as it is the deadliest cancer worldwide. One inflammatory pathway commonly dysregulated in cancer is the metabolism of arachidonic acid (AA) by Cyclooxygenase-2 (COX-2) and microsomal Prostaglandin E Synthase 1 (mPGES-1) into Prostaglandin E2 (PGE2). While researchers have identified PGE2’s pro-tumorigenic functions, the mechanisms governing overexpression of COX-2 and mPGES-1 are incompletely understood. MicroRNAs (miRNAs) are important post-transcriptional regulators commonly dysregulated in cancer. Interestingly, miR-708-5p (miR-708) is predicted to target both COX-2 and mPGES-1. In this study, we show that high miR-708 expression is associated with survival rates in lung squamous cell carcinoma patients. miR-708 also represses PGE2 production by suppressing both COX-2 and mPGES-1 expression in lung cancer cells. miR-708 regulation of COX-2 and mPGES-1 is mediated through targeting of their 3′ untranslated regions (UTRs). Moreover, miR-708 decreases proliferation, survival, and migration of lung cancer cells, which can be partially attributed to miR-708’s inhibition of PGE2 signaling. Lastly, we identify novel miR-708 predicted targets and possible regulators of miR-708 expression in lung cancer. Collectively, these data demonstrate that dysregulated miR-708 expression contributes to exacerbated PGE2 production, leading to an enhanced pro-tumorigenic phenotype in lung cancer cells.

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using in vitro kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from in vitro and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.

Survival impact of multidisciplinary thoracic oncology care in a regional healthcare system.

2004 Background: Much-advocated, the value and impact of multidisciplinary care and planning (MDC) needs greater evidence. We compared non-small cell lung cancer (NSCLC) patient characteristics, treatment patterns and survival in a large community healthcare system spanning 3 US states with some of the highest lung cancer incidence and mortality rates. Methods: We identified MDC patients in the Tumor Registry NSCLC data from 2011-2017. Because the MDC program was located in metropolitan Memphis, we separated non-MDC patients by location of care resulting in 3 cohorts: MDC, non-MDC metropolitan care and non-MDC regional care. Using National Comprehensive Cancer Network guidelines, we categorized treatment by stage as ‘preferred’, ‘appropriate’ (allowable under certain circumstances). We compared demographic and clinical characteristics across cohorts using chi-squared tests and compared survival using Cox regression with Bonferroni adjustment. We repeated survival analysis with propensity matched cohorts. Results: Of 6259 patients, 14% received MDC, 56% metro care and 30% regional care; MDC had the highest rates of African Americans (34% v 28% v 22%), stage I-IIIB (63 v 40 v 50), urban residents (81 v 78 v 20), stage-preferred treatment rates (66 v 57 v 48), stage-appropriate treatment rates (78 v 70 v 63;), and lowest non-treatment rates (6 v 21 v 28). All p<0.001. Compared to MDC, the hazard for death was higher in metro (1.4, 95% confidence interval 1.3-1.6) and regional (1.7, 1.5-1.9); hazards were higher in regional care v metro (1.2, 1.1-1.3); all p<0.001 after adjustment. Results were similar for MDC comparisons after propensity matching with and without adjusting for preferred treatment. No differences in regional and metro cohorts. Conclusions: In this large community-based healthcare system, receipt of MDC for NSCLC was associated with significantly higher rates of guideline-concordant care and survival, providing strong evidence for recommending rigorous implementation of MDC. [Table: see text]

The p53-53BP1-Related Survival of A549 and H1299 Human Lung Cancer Cells after Multifractionated Radiotherapy Demonstrated Different Response to Additional Acute X-ray Exposure.

Radiation therapy is one of the main methods of treating patients with non-small cell lung cancer (NSCLC). However, the resistance of tumor cells to exposure remains the main factor that limits successful therapeutic outcome. To study the molecular/cellular mechanisms of increased resistance of NSCLC to ionizing radiation (IR) exposure, we compared A549 (p53 wild-type) and H1299 (p53-deficient) cells, the two NSCLC cell lines. Using fractionated X-ray irradiation of these cells at a total dose of 60 Gy, we obtained the survived populations and named them A549IR and H1299IR, respectively. Further characterization of these cells showed multiple alterations compared to parental NSCLC cells. The additional 2 Gy exposure led to significant changes in the kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci numbers in A549IR and H1299IR compared to parental NSCLC cells. Whereas A549, A549IR, and H1299 cells demonstrated clear two-component kinetics of DNA double-strand break (DSB) repair, H1299IR showed slower kinetics of γH2AX foci disappearance with the presence of around 50% of the foci 8 h post-IR. The character of H2AX phosphorylation in these cells was pATM-independent. A decrease of residual γH2AX/53BP1 foci number was observed in both A549IR and H1299IR compared to parental cells post-IR at extra doses of 2, 4, and 6 Gy. This process was accompanied with the changes in the proliferation, cell cycle, apoptosis, and the expression of ATP-binding cassette sub-family G member 2 (ABCG2, also designated as CDw338 and the breast cancer resistance protein (BCRP)) protein. Our study provides strong evidence that different DNA repair mechanisms are activated by multifraction radiotherapy (MFR), as well as single-dose IR, and that the enhanced cellular survival after MFR is reliant on both p53 and 53BP1 signaling along with non-homologous end-joining (NHEJ). Our results are of clinical significance as they can guide the choice of the most effective IR regimen by analyzing the expression status of the p53–53BP1 pathway in tumors and thereby maximize therapeutic benefits for the patients while minimizing collateral damage to normal tissue.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

RAC1 Involves in the Radioresistance by Mediating Epithelial-Mesenchymal Transition in Lung Cancer.

Radiation therapy is a common and acceptable approach for lung cancer. Although the benefit of ionizing radiation (IR) is well-established, cancer cells can still survive via pro-survival and metastasis signaling pathways. Ras related C3 botulinum toxin substrate1 (RAC1), a member of Rho family GTPases, plays important roles in cell migration and survival. In the present study, we investigated the effects of RAC1 on the survival of lung cancer cells treated with irradiation. The results showed RAC1 is overexpressed in lung cancer cells and promoted cell proliferation and survival. Furthermore, IR induced RAC1 expression and activity via the activation of PI3K/AKT signaling pathway, and then enhancing cell proliferation, survival, migration and metastasis and increasing levels of epithelial-to-mesenchymal transition (EMT) markers, which facilitated the cell survival and invasive phenotypes. In addition, overexpression of RAC1 attenuated the efficacy of irradiation, while inhibition of RAC1 enhanced sensitivity of irradiation in xenograft tumors in vivo. Collectively, we further found that RAC1 enhanced radioresistance by promoting EMT via targeting the PAK1-LIMK1-Cofilins signaling in lung cancer. Our finding provides the evidences to explore RAC1 as a therapeutic target for radioresistant lung cancer cells.

AMPK-dependent phosphorylation of HDAC8 triggers PGM1 expression to promote lung cancer cell survival under glucose starvation.

Cancer cells undergo metabolic reprogramming to sustain their own survival under an environment of increased energy demand; however, the mechanism by which cancer cells ensure survival under glucose deprivation stressed conditions remains elusive. Here, we show that deprivation of glucose, dramatically activated the glycogen pathway, accompanied by elevated phosphoglucomutase 1 (PGM1) expression. We further identified that AMP-activated protein kinase (AMPK) stimulated PGM1 expression by inducing histone deacetylase 8 (HDAC8) phosphorylation. Moreover, we demonstrated that glucose deprivation-induced AMPK activation stimulated the translocation of HDAC8 from the nucleus to the cytoplasm, consequently disrupting the binding between HDAC8 and histone 3. PGM1 expression was also found to be critical for lung cancer glycolysis, the oxidative pentose phosphate pathway, and oxidative phosphorylation under glucose deprivation conditions, and further led to the aberrant expression of metabolic enzymes involved in glucose metabolism mediated by ERK1/2. Finally, PGM1 was found to be highly expressed in lung cancer tissues from patients, which correlated with a poor prognosis. Taken together, these results revealed that AMPK activation by glucose deprivation leads to enhanced PGM1 expression, an essential component of the metabolic switch, to facilitate cancer progression, suggesting PGM1 as promising anti-cancer treatment targets.

An RNA-binding-protein, NONO governs energy metabolism by regulating NAMPT in lung cancer

The RNA binding proteins (RBPs) have multiple roles in human cancer. However, their molecular target and function have not been clearly identified. Our genomic analysis derived from patients reveals that NONO is a potential oncogenic gene in lung cancer. NONO is highly expressed in lung cancer tissues compared with normal tissues, and its expression has been correlated with the prognosis of lung cancer patients. We found that NONO significantly influences cancer cell proliferation in lung cancer. Gene expression profiles with NONO-depleted cells revealed that the sirtuin signaling pathway is highly correlated with NONO. Thus, NONO-silenced cells caused reduction of the TCA cycle and glycolysis metabolism. We identified that NONO regulated NAMPT, which is a well-known gene involved in sirtuin signaling, and NONO has a significant correlation with NAMPT in lung cancer patients. We propose that NONO modulates energy metabolism by direct interaction with NAMPT and suggest that a functional relationship between NONO and NAMPT contributes to lung cancer cell survival. Targeting the axis can be a promising approach for patient treatment in lung cancer.

Depletion of histone methyltransferase KMT9 inhibits lung cancer cell proliferation by inducing non-apoptotic cell death

BackgroundLung cancer is the leading cause of cancer related death worldwide. Over the past 15 years no major improvement of survival rates could be accomplished. The recently discovered histone methyltransferase KMT9 that acts as epigenetic regulator of prostate tumor growth has now raised hopes of enabling new cancer therapies. In this study, we aimed to identify the function of KMT9 in lung cancer.MethodsWe unraveled the KMT9 transcriptome and proteome in A549 lung adenocarcinoma cells using RNA-Seq and mass spectrometry and linked them with functional cell culture, real-time proliferation and flow cytometry assays.ResultsWe show that KMT9α and -β subunits of KMT9 are expressed in lung cancer tissue and cell lines. Importantly, high levels of KMT9α correlate with poor patient survival. We identified 460 genes that are deregulated at the RNA and protein level upon knock-down of KMT9α in A549 cells. These genes cluster with proliferation, cell cycle and cell death gene sets as well as with subcellular organelles in gene ontology analysis. Knock-down of KMT9α inhibits lung cancer cell proliferation and induces non-apoptotic cell death in A549 cells.ConclusionsThe novel histone methyltransferase KMT9 is crucial for proliferation and survival of lung cancer cells harboring various mutations. Small molecule inhibitors targeting KMT9 therefore should be further examined as potential milestones in modern epigenetic lung cancer therapy.

Colicin N Mediates Apoptosis and Suppresses Integrin-Modulated Survival in Human Lung Cancer Cells.

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5–15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10–15 µM) for 12 h. Moreover, 5–15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin β1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Combination of AAV‑mediated NUPR1 knockdown and trifluoperazine induces premature senescence in human lung adenocarcinoma A549cells in nude mice.

Nuclear protein 1 (NUPR1)/p8, a transcriptional regulator, has the ability to facilitate lung cancer cell survival. Adeno‑associated virus (AAV)‑based vectors are efficient vehicles for gene transfer and expression. In this study, an AAV‑mediated NUPR1 shRNA vector was constructed that effectively inhibited the expression of NUPR1 in a tumor xenograft model derived from lung adenocarcinoma A549cells. Trifluoperazine (TFP), which is an antipsychotic drug, has the ability to bind to NUPR1 and mimic NUPR1 deficiency in cancer cells. It was also found that the combination of TFP and AAV‑mediated NUPR1 shRNA delivery led to significant tumor growth inhibition in nude mice bearing human lung cancer xenografts. Moreover, AAV‑mediated NUPR1 shRNA therapy induced premature senescence invitro and invivo. Collectively, the findings of this study suggest a putative role for the combination of AAV‑NUPR1 shRNA and TFP in lung cancer therapy.

YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.

Targeted inhibition of GRP78 by HA15 promotes apoptosis of lung cancer cells accompanied by ER stress and autophagy.

ABSTRACTThis study investigated the pathophysiological role of GRP78 in the survival of lung cancer cells. Lung cancer patient data from public databases were used to analyze the expression of GRP78 and its influence on prognoses. In vivo, GRP78 protein expression was analyzed in an established urethane-induced lung tumor mouse model. In vitro, the effects of targeted inhibition of GRP78 by HA15 in lung cancer cells were assessed, with cell viability analyzed using a CCK-8 assay, cell proliferation using an EdU assay, apoptosis and cell cycle using flow cytometry, subcellular structure using electron microscopy, and relative mRNA and protein expression using RT-PCR, western blotting or immunofluorescence assays. The results showed that GRP78 was highly expressed in the lung tissue of lung cancer mice model or patients, and was associated with a poor prognosis. After inhibition of GRP78 in lung cancer cells by HA15, cell viability was decreased in a dose- and time-dependent manner, proliferation was suppressed and apoptosis promoted. Unfolded protein response signaling pathway proteins were activated, and the autophagy-related proteins and mRNAs were upregulated. Therefore, targeted inhibition of GRP78 by HA15 promotes apoptosis of lung cancer cells accompanied by ER stress and autophagy.

In vitro antiproliferative activity of cold atmospheric plasma on small-cell lung carcinoma

Background: Cold plasma is shown to inhibit the cancer cell growth. Manipulation of different plasma parameters might have influence on the production of major reactive species which leads to killing of the cancer cells. Antiproliferative activity of cold atmospheric pressure plasma jet was investigated on small-cell lung carcinoma BHY cell line (squamous cell carcinoma) under different in vitro conditions. Methods: A homemade plasma jet was designed and created using pure helium gas. To identify the species created by the plasma jet, optical emission spectroscopy (OES) was employed. Next, the effect of plasma jet was examined on lung cancer cell survival by MTT assay and the effects of main parameters were evaluated on plasma performance. In this favor, various treatment times including 60, 90, 120, 180, and 300 s in combination with different voltages of 5, 11, and 14 kV were investigated, and the results were analyzed at 2, 24, and 48 h after exposure to plasma. Results: Predominant species of OES spectra were O, OH, N2+, and N2. Results of MTT assay indicated a dramatic reduction in cell viabilities in both dose- and time-dependent manners, and more than 75% of cancer cells were died after 48 h at 180 s of plasma treatment. Conclusion: The homemade plasma jet can chiefly contribute to the production of reactive oxygen and nitrogen species (reactive oxygen species and reactive nitrogen species) and can induce apoptosis in small-cell lung carcinoma BHY cell line.

TIPE2 Induced the Proliferation, Survival, and Migration of Lung Cancer Cells Through Modulation of Akt/mTOR/NF-κB Signaling Cascade.

Lung cancer represents the most common cause of cancer deaths in the world, constituting around 11.6% of all new cancer cases and 18.4% of cancer-related deaths. The propensity for early spread, lack of suitable biomarkers for early diagnosis, as well as prognosis and ineffective existing therapies, contribute to the poor survival rate of lung cancer. Therefore, there is an urgent need to develop novel biomarkers for early diagnosis and prognosis which in turn can facilitate newer therapeutic avenues for the management of this aggressive neoplasm. TIPE2 (tumor necrosis factor-α-induced protein 8-like 2), a recently identified cytoplasmic protein, possesses enormous potential in this regard. Immunohistochemical analysis showed that TIPE2 was significantly upregulated in different stages and grades of lung cancer tissues compared to normal lung tissues, implying its involvement in the positive regulation of lung cancer. Further, knockout of TIPE2 resulted in significantly reduced proliferation, survival, and migration of human lung cancer cells through modulation of the Akt/mTOR/NF-κB signaling axis. In addition, knockout of TIPE2 also caused arrest in the S phase of the cell cycle of lung cancer cells. As tobacco is the most predominant risk factor for lung cancer, we therefore evaluated the effect of TIPE2 in tobacco-mediated lung carcinogenesis as well. Our results showed that TIPE2 was involved in nicotine-, nicotine-derived nitrosamine ketone (NNK)-, N-nitrosonornicotine (NNN)-, and benzo[a]pyrene (BaP)-mediated lung cancer through inhibited proliferation, survival, and migration via modulation of nuclear factor kappa B (NF-κB)- and NF-κB-regulated gene products, which are involved in the regulation of diverse processes in lung cancer cells. Taken together, TIPE2 possesses an important role in the development and progression of lung cancer, particularly in tobacco-promoted lung cancer, and hence, specific targeting of it holds an enormous prospect in newer therapeutic interventions in lung cancer. However, these findings need to be validated in the in vivo and clinical settings to fully establish the diagnostic and prognostic importance of TIPE2 against lung cancer.

Cancer-educated mesenchymal stem cells promote the survival of cancer cells at primary and distant metastatic sites via the expansion of bone marrow-derived-PMN-MDSCs

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stromal cells that can differentiate into a variety of cell types. BMSCs are chemotactically guided towards the cancer cells and contribute to the formation of a cancer microenvironment. The homing of BMSCs was affected by various factors. Disseminated tumour cells (DTCs) in distant organs, especially in the bone marrow, are the source of cancer metastasis and cancer relapse. DTC survival is also determined by the microenvironment. Here we aim to elucidate how cancer-educated BMSCs promote the survival of cancer cells at primary tumour sites and distant sites. We highlight the dynamic change by identifying different gene expression signatures in intratumoral BMSCs and in BMSCs that move back in the bone marrow. Intratumoral BMSCs acquire high mobility and displayed immunosuppressive effects. Intratumoral BMSCs that ultimately home to the bone marrow exhibit a strong immunosuppressive function. Cancer-educated BMSCs promote the survival of lung cancer cells via expansion of MDSCs in bone marrow, primary tumour sites and metastatic sites. These Ly6G+ MDSCs suppress proliferation of T cells. CXCL5, nitric oxide and GM-CSF produced by cancer-educated BMSCs contribute to the formation of malignant microenvironments. Treatment with CXCL5 antibody, the iNOS inhibitor 1400w and GM-CSF antibody reduced MDSC expansion in the bone marrow, primary tumour sites and metastatic sites, and promoted the efficiency of PD-L1 antibody. Our study reveals that cancer-educated BMSCs are the component of the niche for primary lung cancer cells and DTCs, and that they can be the target for immunotherapy.

Inhibition of STAT3 signaling induces apoptosis and suppresses growth of lung cancer: good and bad

Signal transducer and activator of transcription 3 (STAT3) modulates a variety of genes involved in the regulation of critical functions, including cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, and immunity. For many cancers, elevated levels of STAT3 signaling have been associated with a poor prognosis and the development of chemotherapy resistance. In this study, we investigated the inhibitory effects of a novel small-molecule inhibitor of STAT3, STX-0119, on the cell viability and survival of human lung cancer cells. STX-0119 inhibited activated STAT3 and the expression of STAT3-regulated oncoproteins such as c-Myc, cyclin D1, and survivin in lung cancer cells. STX-0119 also decreased the amount of STAT3 in the nuclear fraction as well as induced apoptosis of these lung cancer cell lines as evidenced by increases in apoptotic cells (Annexin V positive) and poly (ADP-ribose) polymerase (PARP) cleavage. The efficacy of STX-0119 in a mouse xenograft model was confirmed. However, a hematological side effect, which had not been previously reported, was observed. The level of white blood cells was significantly lowered when treated at the dose at which STX-0119 alone showed a significant tumor-suppressive effect. In conclusion, we suggest that STX-0119 may be a potent therapeutic agent against lung cancer. Consideration of the side effect suggests, it is necessary to study whether low-dose STX-0119 is effective for lung treatment with a combination of classic lung cancer therapeutics.