Resistant Non-small Cell Lung Carcinoma Research Articles

NUAK1-Mediated Phosphorylation of NADK Mitigates ROS Accumulation to Promote Osimertinib Resistance in Non-Small Cell Lung Carcinoma.

Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, is approved as a first-line therapy in advanced non-small cell lung carcinoma (NSCLC) patients with EGFR-activating mutations or the T790M resistance mutation. However, the efficacy of osimertinib is limited due to acquired resistance, highlighting the need to elucidate resistance mechanisms to facilitate the development of improved treatment strategies. Here, we screened for significantly upregulated genes encoding protein kinases in osimertinib-resistant NSCLC cells and identified NUAK1 as a pivotal regulator of osimertinib resistance. NUAK1 was highly expressed in osimertinib-resistant NSCLC and promoted the emergence of osimertinib resistance. Genetic or pharmacological blockade of NUAK1 restored the sensitivity of resistant NSCLC cells to osimertinib in vitro and in vivo. Mechanistically, NUAK1 directly interacted with and phosphorylated NADK at serine 64 (S64), which mitigated osimertinib-induced accumulation of reactive oxygen species (ROS) and contributed to the acquisition of osimertinib resistance in NSCLC. Furthermore, virtual drug screening identified T21195 as an inhibitor of NADK-S64 phosphorylation, and T21195 synergized with osimertinib to reverse acquired resistance by inducing ROS accumulation. Collectively, these findings highlight the role of the NUAK1-NADK axis in governing osimertinib resistance in NSCLC and indicate the potential of targeting this axis as a strategy for circumventing resistance.

The effect of PLK1 inhibitor in Osimertinib resistant NCI-H1975 cell lines.

To investigate the effects of PLK1 inhibitors on Osimertinib-resistant NCI-H1975 cell lines and the anti-tumor effect combined with Osimertinib. Osimertinib resistant NCI-H1975 cell lines were constructed by the methods of concentration-increasing.; The classical tumor pathway inhibitor library compounds were combined with Osimertinib on Osimertinib-resistant cells to screen compounds with synergistic effects with Osimertinib; The gene set enrichment analysis (GSEA) was used to investigate the activations of signaling pathways in Osimertinib-resistant cells; Sulforhodamine B (SRB) staining was used to investigate the inhibitory effect of PLK1 inhibitors on Osimertinib-resistant cells and the anti-tumor effect of PLK1 inhibitors combined with Osimertinib. Osimertinib-resistant cells lines H1975-resistance (resistance index=43.45) were successfully established. The PLK1 inhibitors BI 2536 and GSK461324 have synergistic effect with Osimertinib. Compared with Osimertinib-sensitive cells, PLK1 regulatory pathway and cell cycle pathway were significantly activated in Osimertinib-resistant cells. In a cohort of NSCLC patients with epidermal growth factor receptor mutations treated with Osimertinib, PLK1 mRNA levels were negatively correlated with progression free survival of patients (R=0.62, P<0.05), confirming that excessive activation of PLK1 in NSCLC cells may cause cell resistance to Osimertinib. Further vitro experiments showed that, The IC50 of PLK1 inhibitors BI 6727 and GSK461324 in Osimertinib-resistant cells were lower than those in sensitive ones. Compared to the mono treatment of Osimertinib, PLK1 inhibitors combined with Osimertinib behave significantly stronger effect on the proliferation of Osimertinib-resistant cells. PLK1 inhibitors combined with Osimertinib behave stronger anti-tumor effect to Osimertinib-resistant NSCLC cells and may be used for intervention and treatment of Osimertinib resistance.

Abstract B026: Overcoming KRAS G12C inhibitor resistance with chaperone-mediated protein degrader in NSCLC

Abstract KRAS-G12C targeting inhibitors (G12Cis), such adagrasib and sotorasib, have shown specificity and promising anti-tumor potential in non-small cell lung carcinoma (NSCLC). However, acquired resistance to these inhibitors has recently been observed in the clinic. Mechanisms of resistance to G12Cis are associated with genetic alterations of several pro-growth signaling pathways. A strategy to overcome the multifarious resistance mechanisms to G12Cis that does not require further molecular characterization is needed. To develop an all-round strategy to overcome G12Ci resistance mechanisms, we exposed NCI-H358, NCI-H1373, and MIAPaCa2 cell lines harboring KRAS-G12C mutations to increasing concentrations of sotorasib or adagrasib over 6 months. The resulting resistant cells proliferate normally in the presence of 10 μM sotorasib or adagrasib. Sotorasib-resistant cells designated as NCI-H358S-R, NCI-H1373S-R, and MIAPaCa2S-R remarkably remain sensitive to adagrasib, suggesting but not proving that the resistant cells remain dependent on mutant KRAS for survival. Adagrasib-resistant cells designated as NCI-H358A-R, NCI-H1373A-R, and MIAPaCa2A-R in contrast remain resistant to both sotorasib and adagrasib. Treatment of adagrasib-resistant cells with 500 nM adagrasib attenuated KRAS activity, however, this was compensated by activation of varied receptor tyrosine kinases (RTKs) to restore phospho-ERK levels, a surrogate marker of RAS activity. Moreover, treatment of de novo resistant NSCLC cells lines NCI-H2030, NCI-H1792, and NCI-H23 or NSCLC patient derived organoids (PDOs) with 500 nM adagrasib resulted in sustained phospho-ERK levels through activation of RTKs. These results indicate the need to simultaneously reduce both KRAS G12C and compensating RTK activity to effectively treat NSCLC. The majority of oncogenic RTKs are dependent on the molecular chaperone HSP90 to maintain stability. Therefore, a heterobifunctional chaperone mediated protein (CHAMP) degrader, RNK07421, was designed to specifically target both KRAS G12C and HSP90. RNK07421 induces an artificial ternary complex between KRAS G12C and HSP90, while also inhibiting the activity of both proteins. The RNK07421 coordinated KRAS G12C-HSP90 interaction promotes the proteasome degradation of KRAS G12C likely via HSP90 associated E3-ligases. Treatment of adagrasib resistant NSCLC cells and PDOs with RNK07421 resulted in reduced levels of KRAS G12C and phospho-ERK along with resistance enabling RTKs: EGFR, BRAF and FGFR2. Furthermore, in vivo treatment of resistant NSCLC cells with RNK07421 demonstrated dramatic tumor reduction as compared to G12Ci. Together these observations indicate that the novel mechanisms of action of RNK07421 may provide several advantages to G12Cis and possibly other targeted protein degradation agents to effectively treat KRAS G12C dependent NSCLC. Citation Format: Ines Pulido, Jennifer M. Graeber, Brianna C. Borger, Kevin P. Foley, Weiwen Ying, Chenghao Ying, Jinhua Li, Yaya Wang, Yuetong Sun, Yan Dai, Guoqiang Wang, Thomas L. Prince, Takeshi Shimamura. Overcoming KRAS G12C inhibitor resistance with chaperone-mediated protein degrader in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B026.

Abstract 1088: Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance

Abstract Purpose. Resistance to radiation, both intrinsic and acquired, remains a significant clinical challenge. It is therefore import to identify the underlying molecular and cellular features involved in this resistance. Materials and Methods. We generated genetically-matched counterparts of various degrees of radiation sensitivity in non-small cell lung carcinoma (NSCLC) tumor models to define characteristics of acquired resistance through repeated exposures to conventional X-ray radiation. Results. As assessed by cell viability and clonogenic assays murine Lewis lung carcinoma (LLC) and human A549 cell lines acquired an approximate 1.5 - 2-fold increase in radiation resistance (RR) as compared to its parental counterpart. Morphologically, we found that A549-RR exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its respective counterpart. Since the N/C ratio has been linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 have a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR, as compared to A549. Subsequently, we confirmed that the LLC-RR and A549-RR are also more resistant to radiotherapy than their isogenic-matched counterpart in vivo. Remarkably, we found that the acquired radiation resistance also induced resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevated thiol levels. Decreasing the amount of thiols with buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, sensitized the resistant cells to cisplatin, to levels similar as parental cells. Conclusions. By generating genetically-matched radiation resistant NSCLC tumor models we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for multi-modality treatment regimens with the potential of improving clinical outcomes in the future. Citation Format: Samir V. Jenkins, Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, Ruud P. Dings. Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1088.

Bisdemethoxycurcumin sensitizes the response of cisplatin resistant non-small cell lung carcinoma cell lines by activating apoptosis and autophagy

Lung cancer belongs to the most frequent and deadliest cancer types worldwide, non-small cell lung carcinoma (NSCLC) being the most frequent type. Development of chemoresistance in NSCLC patients is common and responsible for bad outcome. Curcuminoids are naturally occurring substances with prominent cytotoxic effects in different cancer cells. Here we analyzed influence of bisdemethoxycurcumin (BDMC) on phenotype and molecular mechanisms in cisplatin-sensitive NSCLC cell lines (A549 and H460) and their cisplatin-resistant counterparts. NSCLC cell lines were exposed to BDMC and analyzed by cell viability, proliferation, and motility assays, as well as fluorescence-activated cell sorting. Immunoblotting was assessed to detect apoptosis and autophagy. Colony-formation assay and multicellular tumor spheroid model were used to investigate the effects of BDMC. Expression levels of different Hedgehog-pathway genes were determined by RT-qPCR analysis. We identified substantial cytotoxic effects of BDMC on NSCLC cells in general and on cisplatin-resistant NSCLC cells in special. BDMC markedly decreased the cell viability by inducing apoptosis and autophagy in a cell-type specific manner. BDMC emphasized cisplatin-induced cell death and inhibited cell cycle progression of cisplatin-resistant NSCLC cells. Scratch-closure, colony formation, and multicellular spheroid growth in cisplatin-resistant NSCLC cell lines were inhibited by BDMC. Expression profile analyses of different Hedgehog-pathway regulatory genes showed that Gli1, the mean transcriptional regulator of this pathway, was markedly decreased upon the BDMC treatment, this decrement being most prominent in cisplatin-resistant cells. Our data identified BDMC as a potent substance that may be suitable for combined cisplatin-based therapy in cisplatin-resistant subpopulation of NSCLC patients.

Dual targeting of MEK and PI3K effectively controls the proliferation of human EGFR-TKI resistant non-small cell lung carcinoma cell lines with different genetic backgrounds

BackgroundMolecular targeted therapy for non-small cell lung carcinoma (NSCLC) is restricted due to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This study evaluated the effects of dual targeting of MEK and PI3K in human EGFR-TKI resistant NSCLC cell lines.MethodsEGFR-TKI resistant NSCLC cell lines H1975, H460, and A549, with different mutation and amplification status in EGFR, K-RAS, PIK3CA, and MET genes, were treated with a MEK162 (MEK inhibitor) and BKM120 (PI3K inhibitor) combination or a BIBW2992 (EGFR inhibitor) and ARQ197 (MET inhibitor) combination and assayed for cell proliferation, apoptosis, and cell cycle distribution.ResultsDual targeting of MEK and PI3K efficiently inhibited the cell proliferation, induced apoptosis and the G0/G1 cell cycle, and decreased the phosphorylation of ERK1/2, AKT, S6, and 4E-BP1. H460 cells with K-RAS and PIK3CA mutation were most sensitive to MEK162 and BKM120 combinations. H1975 cells with EGFR and PIK3CA mutation and MET amplification were sensitive to BIBW2992 and ARQ197 combinations.ConclusionDual targeting regulated the proliferation of EGFR-TKI-resistant NSCLC cells, especially mutants in K-RAS and PIK3CA that are promising for EGFR-TKI-resistant NSCLC therapeutics.

Aspirin enhances cisplatin sensitivity of resistant non-small cell lung carcinoma stem-like cells by targeting mTOR-Akt axis to repress migration

Conventional chemotherapeutic regimens are unable to prevent metastasis of non-small cell lung carcinoma (NSCLC) thereby leaving cancer incurable. Cancer stem cells (CSCs) are considered to be the origin of this therapeutic limitation. In the present study we report that the migration potential of NSCLCs is linked to its CSC content. While cisplatin alone fails to inhibit the migration of CSC-enriched NSCLC spheroids, in a combination with non-steroidal anti inflammatory drug (NSAID) aspirin retards the same. A search for the underlying mechanism revealed that aspirin pre-treatment abrogates p300 binding both at TATA-box and initiator (INR) regions of mTOR promoter of CSCs, thereby impeding RNA polymerase II binding at those sites and repressing mTOR gene transcription. As a consequence of mTOR down-regulation, Akt is deactivated via dephosphorylation at Ser473 residue thereby activating Gsk3β that in turn causes destabilization of Snail and β-catenin, thus reverting epithelial to mesenchymal transition (EMT). However, alone aspirin fails to hinder migration since it does not inhibit the Integrin/Fak pathway, which is highly activated in NSCLC stem cells. On the other hand, in aspirin pre-treated CSCs, cisplatin stalls migration by hindering the integrin pathway. These results signify the efficacy of aspirin in sensitizing NSCLC stem cells towards the anti-migration effect of cisplatin. Cumulatively, our findings raise the possibility that aspirin might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to impede migration of NSCLC stem cells otherwise. This may consequently lead to the advancement of remedial outcome for the metastatic NSCLCs.

Sulfocoumarins, specific carbonic anhydrase IX and XII inhibitors, interact with cancer multidrug resistant phenotype through pH regulation and reverse P-glycoprotein mediated resistance

New 6-triazolyl-substituted sulfocoumarins were described as potent inhibitors of the transmembrane human carbonic anhydrase isoforms, CAIX and CAXII. These membrane associated enzymes that maintain pH and CO2 homeostasis are involved in cancer progression, invasion, and resistance to therapy. Recently, it was shown that CAXII expression associates with the expression of P-glycoprotein in multidrug resistant cancer cells. CAXII regulates P-glycoprotein activity by maintaining high intracellular pHi. In this study, the activity of three new sulfocoumarins was evaluated in three sensitive and corresponding multidrug resistant cancer cell lines with increased P-glycoprotein expression (non-small cell lung carcinoma, colorectal carcinoma and glioblastoma). Compound 3 showed the highest potential for cancer cell growth inhibition in all tested cell lines. Flow cytometric analyses showed that compound 3 induced intracellular acidification, cell cycle arrest in G2/M phase and necrosis in non-small cell lung carcinoma cells. Compound 3 demonstrated irreversible, concentration- and time-dependent inhibition of P-glycoprotein activity in multidrug resistant non-small cell lung carcinoma cells. The suppression of P-glycoprotein activity was accompanied with increased P-glycoprotein expression suggesting a compensatory mechanism of multidrug resistant cancer cells. In addition, compound 3 was able to sensitize multidrug resistant non-small cell lung carcinoma cells to doxorubicin. Overall, results imply that compound 3 has multidrug resistance modulating effect through intracellular acidification and subsequent inhibition of P-glycoprotein activity.

Peroxisome Proliferator Activated Receptor Gamma Sensitizes Non-small Cell Lung Carcinoma to Gamma Irradiation Induced Apoptosis.

The nuclear receptors known as peroxisome proliferator activated receptor gamma (PPARG) are lipid-activated transcription factors that have emerged as key regulators of inflammation. PPARG ligands have been shown to have an anti-proliferative effect on a variety of cancers. These ligands can induce apoptosis via TP53 (Tumor protein p53) or ERK1/2 (Extracellular signal-regulated kinases 1/2) (EPHB2) pathways. However, the exact mechanism is not known. PPAR, a type II nuclear hormone receptor deserves attention as a selective target for radiotherapy. Our study examines the potential of selective agonism of PPARG for radiation therapy in non-small cell lung carcinoma (NSCLC). We found that the overexpression of PPARG protein as well as its induction using the agonist, rosiglitazone was able to stimulate radiation-induced cell death in otherwise radio resistant NSCLC A549 cell line. This cell death was apoptotic and was found to be BAX (BCL2 associated X) mediated. The treatment also inhibited radiation-induced AKT (Protein Kinase B) phosphorylation. Interestingly, the ionising radiation (IR) induced apoptosis was found to be inversely related to TP53 levels. A relatively significant increase in the levels of radiation induced apoptosis was observed in H1299 cells (TP53 null) under PPARG overexpression condition further supporting the inverse relationship between apoptosis and TP53 levels. The combination of PPARG agonist and radiation was able to induce apoptosis at a radiation dose at which A549 and H1299 are radioresistant, thus confirming the potential of the combinatorial strategy. Taken together, PPARG agonism was found to invigorate the radiosensitising effect and hence its use in combination with radiotherapy is expected to enhance sensitivity in otherwise resistant cancer types.

84P - Dual inhibition of AXL and FN14 sensitizes cisplatin in resistant non-small cell lung carcinoma by inducing higher caspase 3 cleavage through FHIT upregulation, both in vivo and in vitro

84P - Dual inhibition of AXL and FN14 sensitizes cisplatin in resistant non-small cell lung carcinoma by inducing higher caspase 3 cleavage through FHIT upregulation, both in vivo and in vitro

Inhibition of autophagy by andrographolide resensitizes cisplatin-resistant non-small cell lung carcinoma cells via activation of the Akt/mTOR pathway

Resistance to cisplatin is a major obstacle for the success of non-small cell lung cancer therapy. The mechanisms underlying cisplatin resistance are not fully understood. In this study, we found that the increase of basal auotophagy accompanied the development of cisplatin resistance. Meanwhile the blockade of the Akt/mTOR pathway occurred in the process. Inhibition of this pathway was induced by cisplatin treatment in the resistant non-small cell lung carcinoma cells. Andrographolide, a natural diterpenoid, promoted the activation of the Akt/mTOR signaling by downregulating PTEN and suppressed autophagy, which subsequently resensitized the resistant cells to cisplatin-mediated apoptosis. Cisplatin treatment in combination with andrographolide significantly prevented the growth of the resistant cells in vivo. These results highlight the involvement of autophagy in cisplatin-resistance development and suggest that inhibition of autophagy via tuning the Akt/mTOR signaling could be a promising strategy in the therapy for cisplatin-resistant non-small cell lung cancer.

Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to restrain angiogenesis.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.

Diarylheptanoids from the bark of black alder inhibit the growth of sensitive and multi-drug resistant non-small cell lung carcinoma cells

An extended study of minor diarylheptanoids from the bark of black alder has resulted in the isolation of twenty diarylheptanoids, ten of which have not previously been reported (14-18, 20-24). The structures and configurations of all compounds were elucidated by NMR, HRESIMS, UV, IR, and CD. The anti-cancer potency of twenty diarylheptanoids and four previously isolated compounds (7, 10, 12, 13) was investigated in human non-small cell lung carcinoma cell lines (sensitive and multi-drug resistant variants) as well as in normal human keratinocytes. Diarylheptanoids with a p-coumaroyl group, 14 and 18, platyphylloside (1), platyphyllonol-5-O-β-D-xylopyranoside (2), alnuside B (4) and hirsutenone (9) exhibited strong anti-cancer activity, considerably higher than diarylheptanoid curcumin, which served as a positive control. Compounds 4, 9, 14, and 18 displayed significant selectivity towards the cancer cells. Structure/activity analysis of twenty-four closely related diarylheptanoids revealed a high dependence of cytotoxic action on the presence of a carbonyl group at C-3. Substitution of a heptane chain on C-5 and a number of hydroxyl groups in the aromatic rings also emerged as a significant structural feature that influenced their cytotoxic potential.

Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma

Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.

Abstract 971: Synergistic effect of c-Met inhibitor volitinib in combination with EGFR inhibitor Gefitnib on EGFR-TKI resistant NSCLC model HCC827C4R harboring acquired Met gene amplification.

Abstract It is well recognized that non-small cell lung carcinoma (NSCLC) patients with activating EGFR mutations will develop drug resistance after receiving EGFR-tyrosine kinase inhibitors (EGFR-TKI). About 20% of those patients with TKI resistance were identified to harbor Met gene amplification. The aim of this study includes two aspects: a) generating an acquired c-Met amplified EGFR-TKI resistant tumor line in preclinical setting, and b) exploring combination effect of c-met inhibitor volitinib and EGFR inhibitor gefitinib on the new tumor line. Volitinib is a novel, highly potent and selective c-Met inhibitor, currently being evaluated in the phase I clinical trial. HCC827(exon 19 del E746-A750), a human NSCLC cell line, was treated with increasing concentrations of EGFR inhibitor for about 6 months and gradually produced the resistance to EGFR-TKIs, such as gefitinib and erlotinib. One of the subclones, HCC827C4R was isolated and was confirmed to carry Met gene amplification in comparison to parent cell line HCC827. It was not sensitive to volitinib treatment in cell survival assay due to dual activation of EGFR and c-Met pathways. The results from signal pathway study demonstrated that in HCC827C4R cells, volitinib or gefitinib alone only inhibited the phosphorylation of c-Met or EGFR, respectively, but had no effect on the activation of downstream molecules such as Akt and ERK which drive tumor cell proliferation and other cell functions. In contrast, combination of volitinib and gefitinib significantly inhibited phosphorylation of EGFR, c-Met, Akt and ERK in HCC827C4R, and consequently led to a synergistic effect on inhibiting tumor cell growth in vitro. These results were further confirmed in HCC827C4R xenograft model in vivo at clinically relevant doses. Combination of volitinib and gefitinib induced significant tumor regression and displayed synergistic effect compared to treatment by either gefitinib or volitinib alone. Consistent with in vitro results, combination group strongly inhibited the downstream Akt and ERK phosphorylation. In addition, combination treatment was well tolerant and no significant drug-drug exposure interaction was observed. These data indicated that both c-Met and EGFR could contribute to activating downstream signaling pathway and control HCC827C4R cell growth. Blocking either pathway may not be strong enough to stop tumor growth. In conclusion, a cell line with activating EGFR mutation and c-Met gene amplification was generated with resistance to gefitinib and insensitivity to volitinib. The combination treatment with volitinib and an EGFR inhibitor gefitinib was highly effective in vitro and in vivo, suggesting that such combination could provide a safe and effective treatment in clinics for this particular patient population. Citation Format: Feng Zhou, Yongxin Ren, Yumin Cui, Hanyang Chen, Longxian Jiao, Guangxiu Dai, Shiming Fan, Junen Sun, Yongjuan Yu, Yang Sai, Yi Gu, Weiguo Qing, Weiguo Su. Synergistic effect of c-Met inhibitor volitinib in combination with EGFR inhibitor Gefitnib on EGFR-TKI resistant NSCLC model HCC827C4R harboring acquired Met gene amplification. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 971. doi:10.1158/1538-7445.AM2013-971

βArrestin-1 and Mcl-1 Modulate Self-Renewal Growth of Cancer Stem-Like Side-Population Cells in Non-Small Cell Lung Cancer

Side population (SP) cells have been reported to have properties of cancer stem-like cells (CSCs) in non-small cell lung carcinoma (NSCLC), yet their molecular features have not been fully elucidated. Here we show that, NSCLC-SP cells were enriched in G0/G­1 phase of cell cycle, had higher aldehyde dehydrogenase activity as well as higher clonogenic and self-renewing ability compared to main population (MP) cells. Interestingly, SP cells were also able to trans-differentiate into angiogenic tubules in vitro and were highly tumorigenic as compared to MP cells. SP-derived tumors demonstrated the intratumoral heterogeneity comprising of both SP and MP cells, suggesting the self-renewal and differentiation ability of SP cells are manifested in vivo as well. βArrestin-1 (βArr1) is involved in the progression of various cancers including NSCLCs and we find that depletion of βArr1 significantly blocked the SP phenotype; whereas depletion of βArr2 had relatively minor effects. Ectopic expression of βArr1 resulted in increased SP frequency and ABCG2 expression while abrogation of βArr1 expression suppressed the self-renewal growth and expansion of A549 cells. Anti-apoptotic protein Mcl-1 is known to be one of the key regulators of self-renewal of tissue stem cells and is thought to contribute to survival of NSCLC cells. Our experiments show that higher levels of Mcl-1 were expressed in SP cells compared to MP cells at both transcriptional and translational levels. In addition, Obatoclax, a pharmacological inhibitor of Mcl-1, could effectively prevent the self-renewal of both EGFR-inhibitor sensitive and resistant NSCLC cells. In conclusion, our findings suggest that βArr1 and Mcl-1 are involved in the self-renewal and expansion of NSCLC-CSCs and are potential targets for anti-cancer therapy.

Abstract 1906: Mechanisms of resistance to EGFR tyrosine kinase inhibitors involving HER-family ligands and receptors

Abstract Known mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor (EGFR-TKI), including the second mutation of EGFR, T790M, MET amplification, and HGF overexpression, explain approximately 60% of cases, but the remaining mechanisms have not been clarified. To investigate novel mechanisms of acquired resistance to EGFR-TKI, we examined HER-family ligands and HER-family receptor status in non-small cell lung carcinoma (NSCLC) cell lines (n=13). Cell lines were divided into two groups with respect to EGFR-TKI sensitivity: highly sensitive (HCC827, PC-9, HCC2935, and HCC4006) and resistant (H1395, H1666, H596, H1437, A549, H226B, H1299, H460, H1792). We examined expression levels of eleven HER-family ligands_EGF, TGFβ, amphiregulin (AREG), betacellulin (BTC), HB-EGF, epigen (EPGN), epiregulin (EREG), and neuregulin (NRG) 1, NRG2, NRG3, and NRG4_in these cell lines. NRG2, NRG4, and EPGN were expressed at 39.5-, 31.4-, and 6.5-fold higher levels, respectively, in EGFR-TKI primary resistant NSCLC cell lines than in highly sensitive cell lines, whereas TGFA, EGF, and AREG were expressed at 0.22-, 0.3-, and 0.34-fold lower levels. Phosphorylation of EGFR was elevated in sensitive cell lines compared to resistant cell lines, but HER2-4 receptors did not evidence a difference in phsophorylation. These results suggest that the EGFR pathway is activated in sensitive cell lines, and HER2-4, which binds to EPGN, EREG, NRG2, and NRG4, might be activated in resistant cell lines. We prepared cell lines HCC827GR and HCC827ER, derived from HCC827, which acquired resistance to gefitinib or erlotinib, respectively. We analyzed HER-family ligands and receptors using these cell lines. Compared to parent cells, EPGN, EREG, HB-EGF, TGFA, NRG1, and AREG were up-regulated in HCC827GR. Phosphorylation status of EGFR, HER2, and HER3 was not altered, but that of HER4 was increased in HCC827GR. In HCC827ER, EGFR binding ligands AREG, BTC, and EGF were down-regulated, while phosphorylation of EGFR, HER2, and HRE3 was decreased. However, NRG2 was remarkably elevated and HER4 and its downstream STAT5 were phosphorylated. These results indicate that HER4 and its binding ligands may contribute to acquired resistance to EGFR-TKI. Considering these results, it is possible that the HER4-STAT5 pathway might be common to primary and acquired resistance to EGFR-TKI. We continue to investigate its significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1906. doi:1538-7445.AM2012-1906

Phase 1 trial of lipoplatin and gemcitabine as a second‐line chemotherapy in patients with nonsmall cell lung carcinoma

: Lipoplatin is a new liposomal cisplatin that already has been tested in solid tumors, with encouraging results. The purpose of the current study was to determine the maximum tolerated dose (MTD) and the dose-limiting toxicity (DLT) of a 21-day regimen of lipoplatin plus a fixed dose of gemcitabine in patients with refractory or resistant nonsmall cell lung carcinoma (NSCLC) with an Eastern Cooperative Oncology Group (ECOG) performance status of </=2. : The lipoplatin dose was escalated at 100 mg/m(2) by increments of 10 mg/m(2) on Days 1 and 8, with gemcitabine at a dose of 1000 mg/m(2) administered on Days 1 and 8, repeated every 21 days. Hematopoietic growth factors were not allowed. Thirteen patients with advanced stage NSCLC who had been pretreated with platinum combination chemotherapy were enrolled in this phase 1 trial. At least 3 patients were entered at each dose level. : At the fourth dose level, the DLT was reached (grade 3 neutropenia [according to World Health Organization criteria] in 3 of 4 patients 75%]; the fourth patient demonstrated degradation of performance status). Therefore, the third dose level (lipoplatin at a dose of 120 mg/m(2)) was defined as the MTD. At the same dose level, 2 of 4 patients had grade 3 thrombocytopenia. At the fourth dose level, 1 patient achieved a partial response and 1 patient had stable disease. Another patient achieved stable disease at the second dose level. Therefore, the overall disease control rate was 23% (3 of 13 patients). The median overall survival was 29 weeks (range, 4 weeks-59 weeks) and the median time to disease progression was 12 weeks (range, 3 weeks-36 weeks). : The pharmacokinetic profile of the 2 compounds used in the current study are not modified when they are administered according to the schedule evaluated in this trial. When one considers that the patients in the current study had refractory or resistant NSCLC, the authors concluded that the combination of lipoplatin administered at a dose of 120 mg/m(2) and gemcitabine administered at a dose of 1000 mg/m(2) on Days 1 and 8 every 3 weeks needs to be studied further in phase 2 trials. Cancer 2008. (c) 2008 American Cancer Society.

Sulfinosine enhances doxorubicin efficacy through synergism and by reversing multidrug resistance in the human non-small cell lung carcinoma cell line (NCI-H460/R)

A resistant non-small cell lung carcinoma cell line-NSCLC (NCI-H460/R) was established in order to investigate the potential of sulfinosine (SF) to overcome multidrug resistance (MDR). The cytotoxicity of doxorubicin (DOX) in NCI-H460/R cells was enhanced by interaction with SF. SF improved the sensitivity of resistant cells to DOX when NCI-H460/R cells were pretreated with SF. Synergism was accompanied by the accumulation of cells in S and G(2)/M phases. Pretreatment with SF was more potent in improving the sensitivity to DOX than verapamil (VER). The decrease of mdr1 and topo II alpha expression (assessed by RT-PCR), was consistent with the DOX accumulation assay and cell cycle analysis. Also, SF significantly decreased intracellular glutathione (GSH) concentration. These results point to SF as a potential agent of MDR reversal and a valuable drug for improving chemotherapy of NSCLC.