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Despite improvements in therapies available for small subsets of patients affected by non-small cell lung cancer (NSCLC), the chemotherapy drug cisplatin is still one of the most commonly used treatments for advanced NSCLC patients in the absence of oncogenic driver mutations or immune checkpoints. Unfortunately, as in the case of many solid tumors, acquired drug resistance is a common phenomenon in NSCLC and presents a significant clinical challenge for oncologists. In order to study and elucidate the cellular and molecular mechanisms implicated in the development of drug resistance in cancer, the use of isogenic models provides a valuable in vitro tool for investigating novel biomarkers and the identification of potential targetable pathways involved in drug-resistant cancers., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lung cancer is the leading cause of cancer-related deaths worldwide. There are two main subtypes: small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). NSCLC accounts for 85% of lung cancer diagnoses. Early lung cancer very often has no specific symptoms, and many patients present with late stage disease. Despite the various treatments currently available, many patients experience tumor relapse or develop therapeutic resistance, highlighting the need for more effective therapies. The development of immunotherapies has revolutionized the cancer treatment landscape by enhancing the body's own immune system to fight cancer. Natural killer (NK) cells are crucial anti-tumor immune cells, and their exclusion from the tumor microenvironment is associated with poorer survival. It is well established that NK cell frequencies and functions are impaired in NSCLC; thus, placing NK cell-based immunotherapies as a desirable therapeutic concept for this malignancy. Immunotherapies such as checkpoint inhibitors are transforming outcomes for NSCLC. This review explores the current treatment landscape for NSCLC, the role of NK cells and their dysfunction in the cancer setting, the advancement of NK cell therapies, and their future utility in NSCLC.

Objectives: The majority of patients with non-small cell lung cancer (NSCLC) present with advanced stage disease, at which time chemotherapy is usually the most common treatment option. While somewhat effective, patients treated with platinum-based regimens will eventually develop resistance, with others presenting with intrinsic resistance. Multiple pathways have been implicated in chemo-resistance, however the critical underlying mechanisms have yet to be elucidated. The aim of this project was to determine the role of inflammatory mediators in cisplatin-resistance in NSCLC., Materials and Methods: Inflammatory mediator, NF-κB, and its associated pathways were investigated in an isogenic model of cisplatin-resistant NSCLC using age-matched parental (PT) and corresponding cisplatin-resistant (CisR) sublines. Pathways were assessed using mass spectrometry, western blot analysis and qRT-PCR. The cisplatin sensitizing potential of an NF-κB small molecule inhibitor, DHMEQ, was also assessed by means of viability assays and western blot analysis., Results: Proteomic analysis identified dysregulated NF-κB responsive targets in CisR cells when compared to PT cells, with increased NF-κB expression identified in four out of the five NSCLC sub-types examined (CisR versus PT). DHMEQ treatment resulted in reduced NF-κB expression in the presence of cisplatin, and re-sensitized CisR cells to the cytotoxic effects of the drug., Conclusion: This study identified NF-ĸB as a potential therapeutic target in cisplatin-resistant NSCLC. Furthermore, inhibition of NF-ĸB using DHMEQ re-sensitized chemo-resistant cells to cisplatin treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. While partial or complete tumor regression can be achieved in patients, particularly with cisplatin-based strategies, these initial responses are frequently short-lived and are followed by tumor relapse and chemoresistance. Identifying the root of cisplatin resistance in NSCLC and elucidating the mechanism(s) of tumor relapse, is of critical importance in order to determine the point of therapeutic failure, which in turn, will aid the discovery of novel therapeutics, new combination strategies and a strategy to enhance the efficacy of current chemotherapeutics. It has been hypothesized that cancer stem cells (CSCs) may be the initiating factor of resistance. We have previously identified and characterized an aldehyde dehydrogenase 1 CSC subpopulation in cisplatin resistant NSCLC. BBI608 is a small molecule STAT3 inhibitor known to suppress cancer relapse, progression and metastasis. Here, we show that BBI608 can inhibit stemness gene expression, deplete CSCs and overcome cisplatin resistance in NSCLC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The PI3K-mTOR pathway is involved in regulating all hallmarks of cancer, and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. Acquired resistance to PI3K-mTOR inhibition is a major hurdle to overcome in the success of PI3K-mTOR targeted agents. H460, A549, and H1975 resistant cells were generated by prolonged treatment in culture with Apitolisib (GDC-0980), a dual PI3K-mTOR inhibitor over a period of several months, from age-matched parent cells. Resistance was deemed to have developed when a log fold difference in IC50 had been achieved. Resistant cell lines also exhibited resistance to another widely investigated PI3K-mTOR dual inhibitor; Dactolisib (BEZ235). Cell lines were characterised at the level of mRNA (expression array profiling expression of >150 genes), miRNA (expression array profiling of 2100 miRNAs), protein (bottoms-up label-free mass spectrometry) and phosphoprotein (expression array profiling of 84 phospho/total proteins). Key alterations were validated by qPCR and Western blot. H1975 cells were initially most sensitive to Apitolisib (GDC-0980), but developed resistance more quickly than the other cell lines, perhaps due to increased selective pressure from the impressive initial effect. In-depth molecular profiling suggested epithelial-mesenchymal transition (EMT) may play a role in resistance to PI3K-mTOR dual inhibition in NSCLC.

Lung cancer is the leading cause of cancer-related deaths. Most patients develop resistance to platinum within several months of treatment. We investigated whether triggering lysosomal membrane permeabilization (LMP) or suppressing autophagy can restore cisplatin susceptibility in lung cancer with acquired chemoresistance. Cisplatin IC50 in A549Pt (parental) and A549cisR (cisplatin resistant) cells was 13 μM and 47 μM, respectively. Following cisplatin exposure, A549cisR cells failed to elicit an apoptotic response. This was manifested by diminished Annexin-V staining, caspase 3 and 9, BAX and BAK activation in resistant but not in parental cells. Chloroquine preferentially promoted LMP in A549cisR cells, revealed by leakage of FITC-dextran into the cytosol as detected by immunofluorescence microscopy. This was confirmed by increased cytosolic cathepsin D signal on Immunoblot. Cell viability of cisplatin-treated A549cisR cells was decreased when co-treated with chloroquine, corresponding to a combination index below 0.8, suggesting synergism between the two drugs. Notably, chloroquine activated the mitochondrial cell death pathway as indicated by increase in caspase 9 activity. Interestingly, inhibition of lysosomal proteases using E64 conferred cytoprotection against cisplatin and chloroquine co-treatment, suggesting that chloroquine-induced cell death occurred in a cathepsin-mediated mechanism. Likewise, blockage of caspases partially rescued A549cisR cells against the cytotoxicity of cisplatin and chloroquine combination. Cisplatin promoted a dose-dependent autophagic flux induction preferentially in A549cisR cells, as evidenced by a surge in LC3-II/α-tubulin following pre-treatment with E64 and increase in p62 degradation. Compared to untreated cells, cisplatin induced an increase in cyto-ID-loaded autophagosomes in A549cisR cells that was further amplified by chloroquine, pointing toward autophagic flux activation by cisplatin. Interestingly, this effect was less pronounced in A549Pt cells. Blocking autophagy by ATG5 depletion using siRNA markedly enhances susceptibility to cisplatin in A549cisR cells. Taken together, our results underscore the utility of targeting lysosomal function in overcoming acquired cisplatin refractoriness in lung cancer.

Background: The VEGF pathway has become an important therapeutic target in lung cancer, where VEGF has long been established as a potent pro-angiogenic growth factor expressed by many types of tumors. While Bevacizumab (Avastin) has proven successful in increasing the objective tumor response rate and in prolonging progression and overall survival in patients with NSCLC, the survival benefit is however relatively short and the majority of patients eventually relapse. The current use of tyrosine kinase inhibitors alone and in combination with chemotherapy has been underwhelming, highlighting an urgent need for new targeted therapies. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC cells and the role of the Neuropilin receptors in this process., Methods: NSCLC cells were screened for expression of VEGF and its receptors. The effects of recombinant VEGF and its blockade on lung tumor cell proliferation and cell cycle were examined. Phosphorylation of Akt and Erk1/2 proteins was examined by high content analysis and confocal microscopy. The effects of silencing VEGF on cell proliferation and survival signaling were also assessed. A Neuropilin-1 stable-transfected cell line was generated. Cell growth characteristics in addition to pAkt and pErk1/2 signaling were studied in response to VEGF and its blockade. Tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells., Results: Inhibition of the VEGF pathway with anti-VEGF and anti-VEGFR-2 antibodies or siRNA to VEGF, NP1 and NP2 resulted in growth inhibition of NP1 positive tumor cell lines associated with down-regulation of PI3K and MAPK kinase signaling. Stable transfection of NP1 negative cells with NP1 induced proliferation in vitro, which was further enhanced by exogenous VEGF. In vivo, NP1 over-expressing cells significantly increased tumor growth in xenografts compared to controls., Conclusions: Our data demonstrate that VEGF is an autocrine growth factor in NSCLC signaling, at least in part, through NP1. Targeting this VEGF receptor may offer potential as a novel therapeutic approach and also support the evaluation of the role of NP1 as a biomarker predicting sensitivity or resistance to VEGF and VEGFR-targeted therapies in the clinical arena.

Aims: To evaluate the potential therapeutic utility of histone deacetylase inhibitors (HDACi) in targeting VEGF receptors in non-small-cell lung cancer., Materials & Methods: Non-small-cell lung cancer cells were screened for the VEGF receptors at the mRNA and protein levels, while cellular responses to various HDACi were examined., Results: Significant effects on the regulation of the VEGF receptors were observed in response to HDACi. These were associated with decreased secretion of VEGF, decreased cellular proliferation and increased apoptosis which could not be rescued by addition of exogenous recombinant VEGF. Direct remodeling of the VEGFR1 and VEGFR2 promoters was observed. In contrast, HDACi treatments resulted in significant downregulation of the Neuropilin receptors., Conclusion: Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for lung cancer patients in the clinical setting.

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of<15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K-NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K-mTOR inhibition for treatment in the chemoresistance setting in NSCLC.