Non-small Cell Lung Cancer Tumor Xenografts Research Articles

CircSLC25A16 facilitates the development of non-small-cell lung cancer through the miR-335-5p/CISD2 axis.

Non-small-cell lung cancer (NSCLC) is a common malignancy with high morbidity and mortality. Circular RNAs are widely involved in NSCLC progression. However, the mechanism of circSLC25A16 in NSCLC has not been reported. The expressions of circSLC25A16, microRNA-335-5p (miR-335-5p), and CDGSH iron-sulfur domain-containing protein 2 (CISD2) were monitored by quantitative real-time fluorescence polymerase chain reaction. Western blot was also carried out to measure the protein levels of CISD2, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA). For functional analysis, cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell, and wound healing assays were utilized to examine cell proliferation, apoptosis, and migration. Glucose uptake and lactate production were detected using commercial kits. The relationship between miR-335-5p and circSLC25A16 or CISD2 was verified by dual-luciferase reporter and RNA immunoprecipitation assays. Furthermore, tumor xenograft was established to explore the function of circSLC25A16 in vivo. CircSLC25A16 and CISD2 were overexpressed in NSCLC, but miR-335-5p was downregulated. CircSLC25A16 acted as a miR-335-5p sponge, and silencing of circSLC25A16 arrested cell proliferation, migration, and glycolysis, and promoted apoptosis, but these impacts were resumed by miR-335-5p inhibition. CISD2 was a miR-335-5p target, and overexpression of CISD2 abolished the suppressive function of miR-335-5p mimic on the malignant behavior of NSCLC cells. CircSLC25A16 could adsorb miR-335-5p to mediate CISD2 expression. Additionally, silencing circSLC25A16 restrained the growth of NSCLC tumor xenograft in vivo. CircSLC25A16 facilitated NSCLC progression via the miR-335-5p/CISD2 axis, implying that circSLC25A16 may serve as a novel biomarker for NSCLC treatment.

Suppression of JAK2/STAT3 Pathway by Notoginsenoside R1 Reduces Epithelial-Mesenchymal Transition in Non-small Cell Lung Cancer.

It has bene reported that a novel saponin-notoginsenoside R1 (NGR1) possesses strong anti-tumor activities. This study aimed to investigate the role and mechanism of NGR1 in non-small cell lung cancer (NSCLC). NSCLC cell viability, proliferation, migration, and invasiveness were assessed using the ex vivo assays. NSCLC xenograft mouse models were constructed to confirm the role of NGR1 in vivo. Epithelial-mesenchymal transition (EMT)-related proteins and key markers in the JAK2/STAT3 pathway were examined using immunoblotting and immunohistochemistry analyses. NGR1 treatment suppressed NSCLC cell growth ex vivo and in vivo. It also decreased the migratory and invasive capacities of NSCLC cells. Additionally, NGR1 increased E-cadherin expression and reduced N-cadherin, vimentin, and snail expression in TGF-β1-treated NSCLC cells and xenograft tumors. JAK2/STAT3 pathway was inhibited by NGR1. Moreover, a specific inhibitor of JAK2, AG490, or STAT3 silencing significantly enhanced the effects of NGR1 against the EMT process in NSCLC cells. NGR1 restrains EMT process in NSCLC by inactivating JAK2/STAT3 signaling, suggesting the potential of NGR1 in anti-NSCLC therapy.

Abstract 1907: MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins

Abstract cMET is an oncogenic receptor tyrosine kinase overexpressed in multiple types of solid tumors, including non-small cell lung cancer (NSCLC), gastric, head and neck, liver cancer and others. Several cMET-directed therapies including antibody-drug conjugates (ADCs) have been developed that may benefit a subset of patients with tumors that have high cMET expression, MET amplification, or dependency on cMET signaling. MYTX-011 is an investigational, pH-sensitive, vcMMAE-based ADC that is designed to potentially benefit not only patients whose tumors express high levels of cMET, but also a broader set of patients whose tumors express low to moderate levels of cMET. MYTX-011 is engineered to rapidly dissociate from cMET only at the acidic pH of endolysosomes. We have previously demonstrated that MYTX-011 drives increased internalization and cytotoxicity in tumor cells expressing moderate cMET levels compared to a matched parent non-pH sensitive ADC. MYTX-011 also demonstrated markedly superior efficacy in NSCLC xenografts with moderate cMET expression as compared to either the parent ADC or benchmark ADCs representing clinical-stage anti-cMET ADCs with vcMMAE and maytansinoid payloads. MYTX-011 was efficacious in NSCLC PDX models with moderate cMET expression of adenocarcinoma/squamous histology and in heterogeneous cMET-expressing EGFR mutant models. In these studies, we demonstrate that MYTX-011 exerts enhanced efficacy by delivering increased levels of payload to cMET+ tumors compared to the parent ADC. MYTX-011 treatment induced significantly increased levels of phospho-histone H3 (pHH3), a pharmacodynamic marker of MMAE cytotoxicity, in moderate cMET expressing NCI-H1975 NSCLC xenograft tumors compared to the parent ADC. Further, increased levels of free MMAE were detected in tumors from mice treated with MYTX-011 compared to a matched dose of the parent ADC, indicating that the pH dependent binding engineered into MYTX-011 translated to increased payload delivery to tumors in vivo. To explore the potential efficacy of MYTX-011 in other cMET-expressing tumor types, we tested the activity of MYTX-011 in a panel of cell lines derived from gastric and head and neck cancers. MYTX-011 demonstrated increased cMET-dependent cytotoxicity compared to the parent ADC. MYTX-011 was also highly active in cMET+ xenograft models derived from gastric (non-MET amplified), esophageal, and head and neck cancers. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET-expressing malignancies. Citation Format: Deepak Kanojia, William Comb, William Israelsen, Federico Colombo, Lin Nie, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011: A pH-dependent anti-cMET ADC with increased payload delivery in vivo and potent activity against cMET-expressing tumors of various epithelial origins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1907.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Human Non-Small Cell Lung Cancer-Chicken Embryo Chorioallantoic Membrane Tumor Models for Experimental Cancer Treatments.

To promote the preclinical development of new treatments for non-small cell lung cancer (NSCLC), we established NSCLC xenograft tumor assays on the chorioallantoic membrane (CAM) of chicken embryos. Five NSCLC cell lines were compared for tumor take rate, tumor growth, and embryo survival. Two of these, A549 and H460 CAM tumors, were histologically characterized and tested for susceptibility to systemic chemotherapy and gene delivery using viral vectors. All cell lines were efficiently engrafted with minimal effect on embryo survival. The A549 cells formed slowly growing tumors, with a relatively uniform distribution of cancer cells and stroma cells, while the H460 cells formed large tumors containing mostly proliferating cancer cells in a bed of vascularized connective tissue. Tumor growth was inhibited via systemic treatment with Pemetrexed and Cisplatin, a chemotherapy combination that is often used to treat patients with advanced NSCLC. Lentiviral and adenoviral vectors expressing firefly luciferase transduced NSCLC tumors in vivo. The adenovirus vector yielded more than 100-fold higher luminescence intensities after a single administration than could be achieved with multiple lentiviral vector deliveries. The adenovirus vector also transduced CAM tissue and organs of developing embryos. Adenovirus delivery to tumors was 100-10,000-fold more efficient than to embryo organs. In conclusion, established human NSCLC-CAM tumor models provide convenient in vivo assays to rapidly evaluate new cancer therapies, particularly cancer gene therapies.

A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer.

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

Gastrodin destabilizes survivin and overcomes pemetrexed resistance

Survivin is a bifunctional protein that plays crucial roles in tumorigenesis. In the present study, we discovered that the natural product gastrodin suppressed the cell viability and colony formation of non-small cell lung cancer (NSCLC) cell lines A549, HCC827, and H460 in a dose-dependent manner. In addition, gastrodin enhanced the protein levels of cleaved-caspase 3 by activating the endogenous mitochondrial apoptosis pathway. Gastrodin inhibits protein kinase B (Akt)/WEE1/cyclin-dependent kinase 1 (CDK1) signaling to downregulate survivin Thr34 phosphorylation. Survivin Thr34 dephosphorylation caused by gastrodin interfered with the binding of ubiquitin-specific protease 19 (USP19), which eventually destabilized survivin. We revealed that the growth of NSCLC xenograft tumors was markedly suppressed by gastrodin in vivo. Furthermore, gastrodin overcomes pemetrexed resistance in vivo or in vitro. Our results suggest that gastrodin is a potential antitumor agent by reducing survivin in NSCLC.

Targeting oncogenic KRAS in non-small cell lung cancer with EGFR aptamer-conjugated multifunctional RNA nanoparticles

KRAS mutations are one of the most common oncogenic driver mutations in human cancers, including non-small cell lung cancer (NSCLC), and have established roles in cancer pathogenesis and therapeutic resistance. The development of effective inhibitors of mutant KRAS represents a significant challenge. Three-way junction (3WJ)-based multi-functional RNA nanoparticles have the potential to serve as an effective invivo siRNA delivery platform with the ability to enhance tumor targeting specificity and visualize biodistribution through an imaging moiety. Herein, we assembled novel EGFRapt-3WJ-siKRASG12C mutation targeted nanoparticles to target EGFR-expressing human NSCLC harboring a KRASG12C mutation to silence KRASG12C expression in a tumor cell-specific fashion. We found that EGFRapt-3WJ-siKRASG12Cnanoparticles potently depleted cellular KRASG12C expression, resulting in attenuation of downstream MAPK pathway signaling, cell proliferation, migration/invasion ability, and sensitized NSCLC cells to chemoradiotherapy. Invivo, these nanoparticles induced tumor growth inhibition in KRASG12C NSCLC tumor xenografts. Together, this study suggests that the 3WJ pRNA-based platform has the potential to suppress mutant KRAS activity for the treatment of KRAS-driven human cancers, and warrants further development for clinical translation.

Echinocystic acid induces the apoptosis, and inhibits the migration and invasion of non-small cell lung cancer cells.

An increasing amount of evidence has demonstrated the anticancer activity of triterpenes extracted from traditional medicines. Echinocystic acid (EA), a natural triterpene isolated from Eclipta prostrata (L.) L., has previously been shown to exhibit anticancer activity in HepG2 and HL-60 cells. The aim of the present study was to investigate the anticancer activity of EA in non-small cell lung cancer (NSCLC) cells. For this purpose, the viability and proliferation of A549 cells were determined using a Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining. The migratory and invasive ability of the A549 cells were measured using wound healing and Transwell assays. Hoechst staining was also performed to detect the apoptosis of A549 cells. The proliferation of A549 cells and the distributions of different growth phases were determined using a flow cytometer. Western blot analysis was used to detect the expression levels of cyclin D, partitioning defective 3 homolog (Par3), PI3K, Akt, mTOR, Bax, Bcl-2 and caspase-3. EA inhibited the proliferation, and the migratory and invasive abilities of cultured lung carcinoma cells (A549 cells), and induced cell cycle arrest in the G1 phase of the cell cycle. Treatment with EA upregulated Par3 expression and inhibited the PI3K/Akt/mTOR pathway in vitro. In addition, EA treatment inhibited tumor growth, suppressed proliferation and induced the apoptosis of tumor cells in NSCLC tumor xenografts in mice. On the whole, these results suggest that EA may represent a potential therapeutic agent for NSCLC.

IKZF4 acts as a novel tumor suppressor in non–small cell lung cancer by suppressing Notch signaling pathway

Non–small cell lung cancer (NSCLC) is the predominant cause of cancer-related mortality globally, although many clinical efforts have been developed to improve the outcomes. The Ikaros zing-finger family transcription factors (IKZFs) have been proved to play pivotal roles in lymphopoiesis and myeloma progression, but their roles in solid tumors development remain unclear. We performed integrative bioinformatical analysis to determine the dysregulation expression of IKZFs in multiple tumors and the correlation between IKZF4 and NSCLC tumor environment. We showed that IKZFs were dysregulated in multiple tumors and IKZF4 was significantly decreased in NSCLC tissues and cell lines due to promoter hypermethylation. We found that low IKZF4 expression obviously correlated with patients' poor clinical outcome. We revealed that IKZF4 overexpression inhibited NSCLC cell growth, migration and xenograft tumor growth, supporting the inhibitory role of IKZF4 in NSCLC tumorigenesis. Additionally, integrative bioinformatical analysis showed that IKZF4 was involved in NSCLC tumor microenvironment. Mechanically, RNA-seq results showed that IKZF4 forced-expression remarkably suppressed Notch signaling pathway in NSCLC, which was validated by qRT-PCR and immunoblot assays. Moreover, we screened several potential agonists for IKZF4.

LncRNA SNHG7 promotes non-small cell lung cancer progression and cisplatin resistance by inducing autophagic activity.

Cisplatin (DDP) is among the most widely used chemotherapeutic drugs for non-small cell lung cancer (NSCLC), yet the frequent emergence of chemoresistance serves as a major barrier to the treatment of this tumor type. Long non-coding RNAs (lncRNAs) have recently been shown to influence the ability of cells to resist particular chemotherapy drugs. The present study was developed to explore the role of the lncRNA SNHG7 as a regulator of NSCLC cell chemosensitivity. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure SNHG7 expression in NSCLC tissues from patients that were sensitive/resistant to DDP, correlations between SNHG7 expression levels and the patients' clinicopathological characteristics were assessed, and the prognostic relevance of SNHG7 expression was examined via the Kaplan-Meier approach. In addition, SNHG7 expression was assessed in NSCLC cell lines that were DDP-sensitive or -resistant, while western blotting and immunofluorescence staining were employed to detect autophagy-associated protein expression in A549, A549/DDP, HCC827, and HCC827/DDP cells. NSCLC cell chemoresistance was quantified via the Cell Counting Kit-8 (CCK-8) assay approach, and flow cytometry was used to detect the apoptotic death of these tumor cells. The chemosensitivity of xenograft tumors in vivo was further assessed to validate the functional importance of SNHG7 as a regulator of NSCLC DDP resistance. Relative to paracancerous tissues, NSCLC tumors exhibited SNHG7 upregulation, and this lncRNA was further upregulated in DDP-resistant patients compared to chemosensitive patients. Consistently, higher SNHG7 expression levels were correlated with worse patient survival outcomes. DDP-resistant NSCLC cells were also found to exhibit higher levels of SNHG7 expression than chemosensitive cells, and knocking down this lncRNA enhanced the sensitivity of these cells to DDP treatment, resulting in impaired proliferation and higher rates of apoptotic death. Knocking down SNHG7 was also sufficient to suppress microtubule associated protein 1 light chain 3 beta (LC3B) and Beclin1 protein levels and promote p62 upregulation in vitro. The silencing of this lncRNA additionally inhibited the resistance of NSCLC xenograft tumors to DDP treatment in vivo. SNHG7 can promote malignant behaviors and DDP resistance in NSCLC cells at least partly via the induction of autophagic activity.

Transcriptional adaptor 3 influences the proliferative and invasive phenotypes of non-small cell lung cancer cells via regulating EMT.

Transcriptional adaptor 3 (TADA3/ADA3) is a conserved transcriptional co-activator and is dysregulated in many aggressive tumors. However, the role of TADA3 in non-small cell lung cancer (NSCLC) remains unknown. It was previously demonstrated that TADA3 expression correlates with poor prognosis in patients with NSCLC. In the present study, the expression and function of TADA3 were investigated in cells in vitro and in vivo. TADA3 expression was evaluated in clinical specimens and cell lines using reverse transcription-quantitative PCR and western blot analysis. The TADA3 protein level was significantly higher in human NSCLC specimens compared with matched normal tissues. In human NSCLC cell lines, short hairpin RNA-mediated silencing of TADA3 suppressed their proliferative, migratory and invasive abilities in vitro, and delayed G1 to S phase progression through the cell cycle. Consistent with this, TADA3 silencing increased expression of the epithelial marker E-cadherin and reduced expression of the mesenchymal markers, N-cadherin, Vimentin, Snail, and Slug. To verify the effect of TADA3 on tumor formation and growth in vivo, a mouse tumor xenograft model was established. TADA3 silencing slowed the growth of NSCLC tumor xenografts in nude mice, and excised tumors showed a similarly altered pattern of epithelial-mesenchymal transition (EMT) marker expression. The present results demonstrated the significance of TADA3 in regulating the growth and metastasis of NSCLC and may provide a theoretical basis for early diagnosis and targeted therapy of NSCLC.

BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer

BackgroundBrother of regulator of imprinted sites (BORIS) is expressed in most cancers and often associated with short survival and poor prognosis in patients. BORIS inhibits apoptosis and promotes proliferation of cancer cells. However, its mechanism of action has not been elucidated, and there is no known inhibitor of BORIS. MethodsA phage display library was used to find the BORIS inhibitory peptides and BTApep-TAT was identified. The RNA sequencing profile of BTApep-TAT-treated H1299 cells was compared with that of BORIS-knockdown cells. Antitumor activity of BTApep-TAT was evaluated in a non-small cell lung cancer (NSCLC) xenograft mouse model. BTApep-TAT was also used to investigate the post-translational modification (PTM) of BORIS and the role of BORIS in DNA damage repair. Site-directed mutants of BORIS were constructed and used for investigating PTM and the function of BORIS.ResultsBTApep-TAT induced DNA damage in cancer cells and suppressed NSCLC xenograft tumor progression. Investigation of the mechanism of action of BTApep-TAT demonstrated that BORIS underwent ADP ribosylation upon double- or single-strand DNA damage. Substitution of five conserved glutamic acid (E) residues with alanine residues (A) between amino acids (AAs) 198 and 228 of BORIS reduced its ADP ribosylation. Inhibition of ADP ribosylation of BORIS by a site-specific mutation or by BTApep-TAT treatment blocked its interaction with Ku70 and impaired the function of BORIS in DNA damage repair.ConclusionsThe present study identified an inhibitor of BORIS, highlighted the importance of ADP ribosylation of BORIS, and revealed a novel function of BORIS in DNA damage repair. The present work provides a practical method for the future screening or optimization of drugs targeting BORIS.

Anesthetic propofol enhances cisplatin-sensitivity of non-small cell lung cancer cells through N6-methyladenosine-dependently regulating the miR-486-5p/RAP1-NF-κB axis

BackgroundDrug resistance is a considerable challenge for chemotherapy in non-small cell lung cancer (NSCLC). Propofol, a commonly used intravenous anesthetics, has been reported to suppress the malignancy of various cancers. However, the effects of propofol on cisplatin (DDP) sensitivity in NSCLC and its molecular mechanisms have not been clearly clarified yet, and the present study aimed to resolve this problem.MethodsNSCLC cells were co-treated with propofol and DDP, Cell Counting kit-8 assay, colony formation assay and flow cytometry were conducted to test the role of propofol in regulating DDP-resistance in NSCLC. Next, through conducting quantitative real-time polymerase chain reaction, dual-luciferase gene reporter system and western blot, the responsible molecular axis in propofol regulating the DDP sensitivity in NSCLC was uncovered, and the function verification experiments were performed by transfection with the inhibitors or small interfering RNAs of those molecules.ResultsPropofol suppressed cell viability, colony formation ability, tumorigenesis, and promoted cell apoptosis to enhance DDP-sensitivity in NSCLC in vitro and in vivo. Propofol increased miR-486-5p level in NSCLC cells and xenograft tumors tissues in a N6-methyladenosine (m6A)-dependent manner, thus inactivating the Ras-associated protein1 (RAP1)-NF-kappaB (NF-κB) axis. Propofol regulated the miR-486-5p/RAP1-NF-κB axis to improve DDP-sensitivity in NSCLC.ConclusionsTaken together, this study firstly investigates the detailed molecular mechanisms by which propofol enhanced DDP-sensitivity in NSCLC cells, and a novel m6A-dependent miR-486-5p/RAP1-NF-κB axis is identified to be closely associated with the process.

CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

Chemotherapy is the mainstay for the treatment of non-small cell lung cancer (NSCLC). However, NSCLC cells are either intrinsically chemoresistant or rapidly develop therapy resistance. Cancer stem cells (CSCs) are widely recognized as the cell population responsible for resistance to systemic therapies, but the molecular responses of CSCs to chemotherapeutic agents are largely unknown. We identified the embryonic protein CRIPTO in stem cell-enriched spheroid cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC) derived from NSCLC surgical specimens. The CRIPTO-positive population had increased clonogenic capacity and expression of stem cell-related factors. Stemness-related properties were also obtained with forced CRIPTO expression, whereas CRIPTO downregulation resulted in cell cycle blockade and CSCs death. Cell populations positive and negative for CRIPTO expression were interconvertible, and interfering with their reciprocal equilibrium resulted in altered homeostasis of cell expansion both in spheroid cultures and in tumor xenografts. Chemotherapy treatment of NSCLC cells resulted in reduction of cell number followed by increased CRIPTO expression and selective survival of CRIPTO-positive cells. In NSCLC tumor xenografts, chemotherapeutic agents induced partial cell death and tumor stabilization followed by CRIPTO overexpression and tumor progression. Altogether, these findings indicate CRIPTO as a marker of lung CSCs possibly implicated in cancer cell plasticity and post-chemotherapy tumor progression.

Xanthohumol inhibits non-small cell lung cancer by activating PUMA-mediated apoptosis

Deregulation of apoptosis signaling is an important feature of cancer cells and plays an essential role in tumorigenesis. Xanthohumol is an active ingredient in Traditional Chinese Medicines Hops (Humulus lupulus L.). Recently studies have shown the profound anti-tumor activities of Xanthohumol in multiple cancer models. However, its potency in non-small cell lung cancer (NSCLC) and the underlying mechanisms are still elusive. Here, we have investigated the potency of Xanthohumol against NSCLC cells in vitro and xenograft mouse models. Xanthohumol suppressed cell viability, colony formation and induced apoptosis in A549, H520, and H358 cells. Xanthohumol activated mitochondrial apoptosis through upregulation of (p53-upregulated modulator of apoptosis) PUMA expression. After Xanthohumol treatment, the Akt activity was inhibited, which resulted in dephosphorylation of FOXO3a and PUMA induction. Silent PUMA or FOXO3a impaired Xanthohumol-induced apoptosis in NSCLC cells. In nude mice, Xanthohumol administration suppressed NSCLC xenograft tumor growth and increased PUMA expression in tumor tissues. Briefly, our studies revealed a novel mechanism by which Xanthohumol exerted its anti-tumor activity in a PUMA-dependent manner in NSCLC cells.

Anticancer activity and metabolic profile alterations by ortho-topolin riboside in in vitro and in vivo models of non-small cell lung cancer.

Lung cancer has the highest incidence and mortality rates among all types of cancer worldwide, and 80%-85% of patients with lung cancer are diagnosed with non-small cell lung cancer (NSCLC), which has 5-year survival rate of only 5% at advanced stages. Development of new therapeutic agents and strategies is required to enhance the treatment efficiency in patients with NSCLC. Metabolic alterations and anticancer effects of plant hormones and their derivatives have not been investigated in NSCLC in vitro and in vivo. The present study investigated the cytotoxic effects of 11 plant hormones and their derivatives against NSCLC cell lines; ortho-topolin riboside (oTR) showed the highest cytotoxicity among all tested compounds against NSCLC cells. Alteration of metabolites and lipids was investigated using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry in oTR-treated NSCLC cells and a xenograft mouse model. oTR reduced amino acid and pyrimidine synthesis in NSCLC cells and xenograft tumors. Moreover, oTR reduced glycolytic function and decreased mitochondrial respiration function by inhibiting glutamine and fatty acid oxidation. Increased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species suggested that oTR might act as a fatty acid oxidation inhibitor. In addition, the increased level of phosphatidylserine species implied that phosphatidylserine-mediated apoptosis occurred in oTR-treated NSCLC cells and xenograft tumor. The antiproliferative and apoptotic effects of oTR were mediated by the reduced p-ERK and p-AKT levels and increased cleaved Caspase-3levels, respectively. This is the first study to investigate the metabolic alterations and anticancer activity of oTR in in vitro and in vivo models of NSCLC. Our results provide basis for the development of oTR-based therapeutic agent for patients with NSCLC.

Heme Sequestration as an Effective Strategy for the Suppression of Tumor Growth and Progression.

Heme is an essential nutritional, metabolic, and signaling molecule in living organisms. Pathogenic microbes extract heme from hosts to obtain metallonutrient, while heme fuels mitochondrial respiration and ATP generation in lung tumor cells. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bind to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non-small cell lung cancer (NSCLC) cells, while their effects on nontumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. IHC, along with data from Western blotting and functional assays, revealed that HeSPs reduced the levels of key proteins involved in heme uptake, as well as the consumption of major fuels for tumor cells, glucose, and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.

Deubiquitinase USP5 promotes non-small cell lung cancer cell proliferation by stabilizing cyclin D1

BackgroundCyclin D1 (CCND1) is overexpressed in non-small cell lung cancer (NSCLC) and contributes to its tumorigenesis and progression. Accumulating evidence shows that ubiquitin-specific protease 5 (USP5), an important member of the USP family, acts as a tumor promoter by deubiquitinating and stabilizing oncoproteins. However, neither the mechanism for dysregulated turnover of CCND1 protein nor the association of CCND1 with USP5 in NSCLC is well understood.MethodsThe association of USP5 with CCND1 in human NSCLC cells and clinical tissues was determined by immunoprecipitation/immunoblotting, immunohistochemistry (IHC), and The Cancer Genome Atlas database analyses. The effect of USP5 knockdown or overexpression on NSCLC cell proliferation in vitro was assessed by Cell Counting Kit-8, flow cytometry-based cell cycle, and colony formation assays. The effect of the USP5 inhibitor EOAI3402143 (G9) on NSCLC proliferation in vitro was analyzed by CCK-8 assay. The effect of G9 on NSCLC xenograft tumor growth was also examined in vivo, using athymic BALB/c nude mice.ResultsUSP5 physically bound to CCND1 and decreased its polyubiquitination level, thereby stabilizing CCND1 protein. This USP5-CCND1 axis promoted NSCLC cell proliferation and colony formation. Further, knockdown of USP5 led to CCND1 degradation and cell cycle arrest in NSCLC cells. Importantly, this tumor-suppressive effect elicited by USP5 knockdown in NSCLC cells was validated in vitro and in vivo through chemical inhibition of USP5 activity using G9. Consistently, G9 downregulated the protein levels of CCND1 in NSCLC cells and xenograft tumor tissues. Also, the expression level of USP5 was positively associated with the protein level of CCND1 in human clinical NSCLC tissues.ConclusionsThis study has provided the first evidence that CCND1 is a novel substrate of USP5. The USP5-CCND1 axis could be a potential target for the treatment of NSCLC.

Targeting c-Myc to Overcome Acquired Resistance of EGFR Mutant NSCLC Cells to the Third-Generation EGFR Tyrosine Kinase Inhibitor, Osimertinib.

Osimertinib (AZD9291 or TAGRISSO) is a promising and approved third-generation EGFR tyrosine kinase inhibitor (TKI) for treating patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, the inevitable emergence of acquired resistance limits its long-term efficacy. A fuller understanding of the mechanism of action of osimertinib and its linkage to acquired resistance will enable the development of more efficacious therapeutic strategies. Consequently, we have identified a novel connection between osimertinib or other EGFR-TKIs and c-Myc. Osimertinib rapidly and sustainably decreased c-Myc levels primarily via enhancing protein degradation in EGFR-mutant (EGFRm) NSCLC cell lines and xenograft tumors. c-Myc levels were substantially elevated in different EGFRm NSCLC cell lines with acquired resistance to osimertinib in comparison with their corresponding parental cell lines and could not be reduced any further by osimertinib. Consistently, c-Myc levels were elevated in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment compared with their corresponding untreated baseline c-Myc levels. Suppression of c-Myc through knockdown or pharmacologic targeting with BET inhibitors restored the response of resistant cell lines to osimertinib. These findings indicate that c-Myc modulation mediates the therapeutic efficacy of osimertinib and the development of osimertinib acquired resistance. Furthermore, they establish c-Myc as a potential therapeutic target and warrant clinical testing of BET inhibition as a potential strategy to overcome acquired resistance to osimertinib or other EGFR inhibitors. SIGNIFICANCE: This study demonstrates a critical role of c-Myc modulation in mediating therapeutic efficacy of osimertinib including osimertinib acquired resistance and suggests targeting c-Myc as a potential strategy to overcome osimertinib acquired resistance.