KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination.

BACKGROUND: KRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions. METHODS: Cell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model. RESULTS: Our research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression. CONCLUSIONS: We posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

Coregulation of pathways in lung cancer patients with EGFR mutation: therapeutic opportunities.

Epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma are a frequent class of driver mutations. Single EGFR tyrosine kinase inhibitor (TKI) provides substantial clinical benefit, but almost nil radiographic complete responses. Patients invariably progress, although survival can reach several years with post-treatment therapies, including EGFR TKIs, chemotherapy or other procedures. Endeavours have been clinically oriented to manage the acquisition of EGFR TKI-resistant mutations; however, basic principles on cancer evolution have not been considered in clinical trials. For years, evidence has displayed rapidly adaptive mechanisms of resistance to selective monotherapy, posing several dilemmas for the practitioner. Strict adherence to non-small cell lung cancer (NSCLC) guidelines is not always practical for addressing the clinical progression that EGFR-mutant lung adenocarcinoma patients suffer. The purpose of this review is to highlight regulatory mechanisms and signalling pathways that cause therapy-induced resistance to EGFR TKIs. It suggests combinatorial therapies that target EGFR, as well as potential mechanisms underlying EGFR-mutant NSCLC, alerting the reader to clinical opportunities that may lead to a deeper and more durable response. Molecular reprogramming contributes to EGFR TKI resistance, and the compiled information is relevant in understanding the development of new combined targeted strategies in EGFR-mutant NSCLC.

Targeting PKCι-PAK1 signaling pathways in EGFR and KRAS mutant adenocarcinoma and lung squamous cell carcinoma.

INTRODUCTION: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models. METHODS: The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models. RESULTS: The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment. CONCLUSIONS: Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

Nuclear IGF-1R predicts chemotherapy and targeted therapy resistance in metastatic colorectal cancer.

BACKGROUND: Although chemotherapy is the cornerstone treatment for patients with metastatic colorectal cancer (mCRC), acquired chemoresistance is common and constitutes the main reason for treatment failure. Monoclonal antibodies against insulin-like growth factor-1 receptor (IGF-1R) have been tested in pre-treated mCRC patients, but results have been largely deceiving. METHODS: We analysed time to progression, overall survival, and the mutational status of RAS, BRAF and nuclear p-IGF-1R expression by immunohistochemistry, in 470 metastatic CRC patients. The effect of IGF-1R activation and distribution was also assessed using cellular models of CRC and RNAi for functional validation. RESULTS: Nuclear IGF-1R increased in metastatic tumours compared to paired untreated primary tumours, and significantly correlated with poor overall survival in mCRC patients. In vitro, chemo-resistant cell lines presented significantly higher levels of IGF-1R expression within the nuclear compartment, and PIAS3, a protein implicated also in the sumoylation process of intranuclear proteins, contributed to IGF-1R nuclear sequestration, highlighting the essential role of PIAS3 in this process. Intriguingly, we observed that ganitumab, an IGF-1R blocking-antibody used in several clinical trials, and dasatinib, an SRC inhibitor, increased the nuclear localisation of IGF-1R. CONCLUSIONS: Our study demonstrates that IGF-1R nuclear location might lead to chemotherapy and targeted agent resistance.

LoVo colon cancer cells resistant to oxaliplatin overexpress c-MET and VEGFR-1 and respond to VEGF with dephosphorylation of c-MET.

Oxaliplatin-resistant LoVo colon cancer cells overexpressing c-MET and VEGFR-1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c-MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin-resistant cells (LoVoR) overexpress and phosphorylate c-MET, upregulate the expression of transmembrane and soluble VEGFR-1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, ß-catenin-TCF4 and E-cadherin. While c-MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c-MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c-MET by VEGF is mediated by VEGFR-1, since phosphorylation of c-MET in the presence of VEGF is restored after silencing VEGFR-1. Dephosphorylation of c-MET by VEGF suggests that tumors coexpressing VEGFR-1 and c-MET may activate c-MET as a result of anti-VEGF therapy.

KRAS G12C-mutant driven non-small cell lung cancer (NSCLC).

KRAS G12C mutations in non-small cell lung cancer (NSCLC) partially respond to KRAS G12C covalent inhibitors. However, early adaptive resistance occurs due to rewiring of signaling pathways, activating receptor tyrosine kinases, primarily EGFR, but also MET and ligands. Evidence indicates that treatment with KRAS G12C inhibitors (sotorasib) triggers the MRAS:SHOC2:PP1C trimeric complex. Activation of MRAS occurs from alterations in the Scribble and Hippo-dependent pathways, leading to YAP activation. Other mechanisms that involve STAT3 signaling are intertwined with the activation of MRAS. The high-resolution MRAS:SHOC2:PP1C crystallization structure allows in silico analysis for drug development. Activation of MRAS:SHOC2:PP1C is primarily Scribble-driven and downregulated by HUWE1. The reactivation of the MRAS complex is carried out by valosin containing protein (VCP). Exploring these pathways as therapeutic targets and their impact on different chemotherapeutic agents (carboplatin, paclitaxel) is crucial. Comutations in STK11/LKB1 often co-occur with KRAS G12C, jeopardizing the effect of immune checkpoint (anti-PD1/PDL1) inhibitors.

Vaccine antibodies against a synthetic epidermal growth factor variant enhance the antitumor effects of inhibitors targeting the MAPK/ERK and PI3K/Akt pathways.

BACKGROUND: The EGFR pathway is involved in intrinsic and acquired resistance to a wide variety of targeted therapies in cancer. Vaccination represents an alternative to the administration of anti-EGFR monoclonal antibodies, such as cetuximab or panitumumab. Here, we tested if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) could potentiate the activity of drugs targeting the ERK/MAPK and PI3K/Akt pathways. METHODS: Non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma cell lines harboring KRAS, NRAS, BRAF and PIK3CA mutations were used. Anti-EGF VacAbs were obtained by immunizing rabbits with a fusion protein containing a synthetic, highly mutated variant of human EGF. Cell viability was determined by MTT, total and phosphorylated proteins by Western blotting, cell cycle distribution and cell death by flow cytometry and emergence of resistance by microscopic examination in low density cultures. RESULTS: Anti-EGF VacAbs potentiated the antiproliferative effects of MEK, KRAS G12C, BRAF, PI3K and Akt inhibitors in KRAS, NRAS, BRAF and PIK3CA mutant cells and delayed the appearance of resistant clones in vitro. The effects of anti-EGF VacAbs were comparable or superior to those of panitumumab and cetuximab. The combination of anti-EGF VacAbs with the targeted inhibitors effectively suppressed EGFR downstream pathways and sera from patients immunized with an anti-EGF vaccine also blocked activation of EGFR effectors. CONCLUSIONS: Anti-EGF VacAbs enhance the antiproliferative effects of drugs targeting the ERK/MAPK and PIK3CA/Akt pathways. Our data provide a rationale for clinical trials testing anti-EGF vaccination combined with inhibitors selected according to the patient's genetic profile.

HMGB1 Expression Levels Correlate with Response to Immunotherapy in Non-Small Cell Lung Cancer.

Purpose: High-mobility group box 1 protein (HMGB1) is subject to exportin 1 (XPO1)-dependent nuclear export, and it is involved in functions implicated in resistance to immunotherapy. We investigated whether HMGB1 mRNA expression was associated with response to immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). Patients and Methods: RNA was isolated from pretreatment biopsies of patients with advanced NSCLC treated with ICI. Gene expression analysis of several genes, including HMGB1, was conducted using the NanoString Counter analysis system (PanCancer Immune Profiling Panel). Western blotting analysis and cell viability assays in EGFR and KRAS mutant cell lines were carried out. Evaluation of the antitumoral effect of ICI in combination with XPO1 blocker (selinexor) and trametinib was determined in a murine Lewis lung carcinoma model. Results: HMGB1 mRNA levels in NSCLC patients treated with ICI correlated with progression-free survival (PFS) (median PFS 9.0 versus 18.0 months, P=0.008, hazard ratio=0.30 in high versus low HMGB1). After TNF-α stimulation, HMGB1 accumulates in the cytoplasm of PC9 cells, but this accumulation can be prevented by using selinexor or antiretroviral drugs. Erlotinib or osimertinib with selinexor in EGFR-mutant cells and trametinib plus selinexor in KRAS mutant abolish tumor cell proliferation. Selinexor with a PD-1 inhibitor with or without trametinib abrogates the tumor growth in the murine Lewis lung cancer model. Conclusion: An in-depth exploration of the functions of HMGB1 mRNA and protein is expected to uncover new potential targets and provide a basis for treating metastatic NSCLC in combination with ICI.

Nuclear factor-kappa B and interleukin-6 related docetaxel resistance in castration-resistant prostate cancer.

BACKGROUND: Previous work showed that the NF-κB survival pathway is activated by docetaxel (D) and contributes to D resistance in prostate cancer. In this study we aimed to investigate the dynamics of the relationship between NF-κB and IL-6 in the shift from D-naive castration-resistant prostate cancer (CRPC) to D-resistance in patients and cell lines. METHODS: CRPC tumor samples were tested for NF-κB/p65 and IL-6 by immunohistochemistry. CRPC patients treated with D were also tested for serum IL-6 (ELISA). Two D-resistant cell lines, PC-3R and DU-145R, derived from the CRPC cells PC-3 and DU-145, respectively, were tested for NF-κB activation (EMSA), NF-κB-related genes expression (RT-PCR), NF-κB inhibition (p65 siRNA) and IL-6 and IL-8 soluble levels (ELISA). RESULTS: In CRPC patients treated with D (n = 72), pre-treatment IL-6 level correlated with nuclear NF-κB/p65 tumor staining and response to D, and was an independent prognostic factor for overall survival. However, IL-6 level changes under treatment did not correlate with clinical outcome. In PC-3 and DU-145 parental CRPC cells, as well as in D-resistant counterparts, D treatment induced NF-κB activation. In fact, NF-κB inhibition was sufficient to re-sensitize DU-145R cells to D. Despite enhanced NF-κB activity, IL-6 secretion in D-resistant cell lines was reduced and not induced by D treatment. The same occurred with IL-8 cytokine. CONCLUSIONS: These preclinical and clinical results support a role of NF-κB and IL-6 in the resistance to D in CRPC, and support the investigation of targeted therapies to enhance the antitumor activity of D in this patient population.

The role of biomarkers in stage III non-small cell lung cancer.

INTRODUCTION: Stage III non-small cell lung cancer (NSCLC) is a composite of the regional spread of lung cancer with different levels of potential lymph node involvement and tumor size that often deem the stage at time of diagnosis to be unresectable and suitable for chemoradiation plus consolidation immunotherapy with durvalumab for 12 months. Chemoradiation plus durvalumab consolidation yielded a landmark 49.2% 5-year overall survival in unresectable NSCLC. AREAS COVERED: Sub-optimal results lead us to focus on the mechanisms of resistance responsible for intractability in a significant proportion of cases that fail with chemoradiation and immunotherapy. In stage III NSCLC it is opportune to explore the accumulated evidence on ferroptosis resistance that can lead to cancer progression and metastasis. Strong data shows that three anti-ferroptosis pathways are principally involved in resistance to chemotherapy, radiation, and immunotherapy. EXPERT OPINION: Because a large part of stage III NSCLCs is resistant to chemoradiation and durvalumab consolidation, a ferroptosis-based therapeutic approach, combined with standard-of-care therapy, can lead to improved clinical outcomes in patients diagnosed with stage III and possibly stage IV NSCLCs.

Biological insights in non-small cell lung cancer.

Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress. Several tumor types, including non-small cell lung cancer (NSCLC), upregulate the system xc- cystine/glutamate antiporter (xCT) through overexpression of the cystine transporter SLC7A11, thus sustaining intracellular cysteine levels to support glutathione synthesis. Nuclear factor erythroid 2-related factor 2 (NRF2) serves as a master regulator of oxidative stress resistance by regulating SLC7A11, whereas Kelch-like ECH-associated protein (KEAP1) acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2. Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC. Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress. Disruptions in cystine availability lead to iron-dependent lipid peroxidation, thus resulting in a type of cell death called ferroptosis. Pharmacologic inhibitors of xCT (either SLC7A11 or GPX4) induce ferroptosis of NSCLC cells and other tumor types. When cystine uptake is impaired, the intracellular cysteine pool can be sustained by the transsulfuration pathway, which is catalyzed by cystathionine-B-synthase (CBS) and cystathionine g-lyase (CSE). The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8+ T cell function and evasion of immunotherapy, diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions. Pyroptosis is a previously unrecognized form of regulated cell death. In NSCLCs driven by EGFR, ALK, or KRAS, selective inhibitors induce pyroptotic cell death as well as apoptosis. After targeted therapy, the mitochondrial intrinsic apoptotic pathway is activated, thus leading to the cleavage and activation of caspase-3. Consequently, gasdermin E is activated, thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis (indicated by characteristic cell membrane ballooning). Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.

Anti-EGF nanobodies enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer (NSCLC) cellular models.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that is effective against the EGFR T790M mutation in patients with advanced non-small-cell lung cancer (NSCLC). However, acquired resistance appears invariably due to several mechanisms. The strategy of using EGF-targeted nanobodies (Nbs) to block the initial step of the EGFR pathway constitutes a new research area. Nbs offer several advantages compared to traditional mAbs, such as their reduced size, increased stability, and tissue penetration, which provide key advantages for targeting soluble tumoral growth factors. In this study we investigated the efficacy of anti-EGF Nbs to reduce Osimertinib resistance. Two anti-EGF Nbs, generated in our laboratory, were shown to inhibit cell viability and colony formation in PC9 and PC9-derived osimertinib-resistant cell lines. The combination of these Nbs with osimertinib improved the antitumor efficacy of this EGFR-TKI in cell viability and colony formation experiments. In a mechanistic study of the EGFR pathway, the combination treatment dampened the activation of downstream proteins such as Akt and Erk1/2 MAP kinases. In addition, it increased cellular apoptosis and decreased the expression of Hes1, a cancer stem cell marker involved in metastasis and osimertinib resistance. We conclude that the addition of anti-EGF nanobodies enhances the antitumor properties of osimertinib, thus representing a potentially effective strategy for NSCLC patients.

Anti-epidermal growth factor vaccine antibodies increase the antitumor activity of kinase inhibitors in ALK and RET rearranged lung cancer cells.

Advanced NSCLC patients harboring EML4-ALK and CCDC6-RET rearrangements derive benefit from treatment with ALK and RET TKIs but not immune checkpoint inhibitors. New immunotherapeutic approaches, such as immunization against growth factors, can be of particular interest for combination treatment in these patients. Here, we investigated the effects of anti-EGF antibodies generated by vaccination (anti-EGF VacAbs), TKIs and combinations in EML4-ALK and CCDC6-RET NSCLC cell lines. We found that EGF and tumor growth factor alpha (TGFα) significantly decreased the antiproliferative activity of the RET inhibitor BLU-667 in CCDC6-RET cells and brigatinib, alectinib and crizotinib in EML4-ALK translocated cells. The addition of anti-EGF VacAbs reversed the effects of EGF and TGFα, potentiated the antitumor effects of the kinase inhibitors and delayed the appearance in vitro of resistant clones. Western blotting demonstrated that the combination of anti-EGF VacAbs with ALK or RET TKIs effectively suppressed EGFR downstream pathways in EML4-ALK translocated and CCDC6-RET cells, respectively. In conclusion, anti-EGF VacAbs significantly increased the antitumor activity of TKIs in ALK and RET-positive cell lines. Clinical trials of an EGF vaccine in combination with ALK and RET TKIs are warranted.

A narrative review of the migration and invasion features of non-small cell lung cancer cells upon xenobiotic exposure: insights from in vitro studies.

Lung cancer (LC) is the leading cause of cancer deaths worldwide, being non-small lung cancer (NSCLC) sub-types the most prevalent. Since most LC cases are only detected during the last stage of the disease the high mortality rate is strongly associated with metastases. For this reason, the migratory and invasive capacity of these cancer cells as well as the mechanisms involved have long been studied to uncover novel strategies to prevent metastases and improve the patients' prognosis. This narrative review provides an overview of the main in vitro migration and invasion assays employed in NSCLC research. While several methods have been developed, experiments using conventional cell culture models prevailed, specifically the wound-healing and the transwell migration and invasion assays. Moreover, it is provided herewith a summary of the available information concerning chemical contaminants that may promote the migratory/invasive properties of NSCLC cells in vitro, shedding some light on possible LC risk factors. Most of the reported agents with pro-migration/invasion effects derive from cigarette smoking [e.g., Benzo(a)pyrene and cadmium] and air pollution. This review further presents several studies in which different dietary/plant-derived compounds demonstrated to impair migration/invasion processes in NSCLC cells in vitro. These chemicals that have been proposed as anti-migratory consisted mainly of natural bioactive substances, including polyphenols non-flavonoids, flavonoids, bibenzyls, terpenes, alkaloids, and steroids. Some of these compounds may eventually represent novel therapeutic strategies to be considered in the future to prevent metastasis formation in LC, which highlights the need for additional in vitro methodologies that more closely resemble the in vivo tumor microenvironment and cancer cell interactions. These studies along with adequate in vivo models should be further explored as proof of concept for the most promising compounds.

Src-Homology 2 Domain-Containing Phosphatase 2 in Resected EGFR Mutation-Positive Lung Adenocarcinoma.

INTRODUCTION: EGFR mutation-positive lung adenocarcinoma (LUAD) displays impaired phosphorylation of ERK and Src-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. We hypothesize that SHP2 expression could be predictive in patients positive with resected EGFR mutation versus patients with EGFR wild-type LUAD. METHODS: We examined resected LUAD cases from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1, and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction. The activity of SHP2 inhibitors plus erlotinib were tested in EGFR-mutant cell lines and analyzed by cell viability assay, Western blot, and immunofluorescence. RESULTS: A total of 50 of 100 EGFR mutation-positive LUADs relapsed, among them, patients with higher SHP2 mRNA expression revealed shorter progression-free survival, in comparison with those having low SHP2 mRNA (hazard ratio: 1.83; 95% confidence interval: 1.05-3.23; p = 0.0329). However, SHP2 was not associated with prognosis in the remaining 167 patients with wild-type EGFR. In EGFR-mutant cell lines, the combination of SHP099 or RMC-4550 (SHP2 inhibitors) with erlotinib revealed synergism via abrogation of phosphorylated AKT (S473) and ERK1/2 (T202/Y204). Although erlotinib translocates phosphorylated SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocates SHP2 into the cytoplasm membrane, limiting AKT and ERK1/2 activation. CONCLUSIONS: Elevated SHP2 mRNA levels are associated with recurrence in resected EGFR mutation-positive LUADs, but not in EGFR wild-type. EGFR tyrosine kinase inhibitors can enhance SHP2 activation, hindering adjuvant therapy. SHP2 inhibitors could improve the benefit of adjuvant therapy in EGFR mutation-positive LUADs.

SRC and PIM1 as potential co-targets to overcome resistance in MET deregulated non-small cell lung cancer.

BACKGROUND: The role of MET alterations in non-small cell lung cancer (NSCLC) is increasing and several targeted agents are under evaluation. MET exon 14 skipping mutations and MET amplifications are associated with potential sensitivity to MET inhibition, though resistance mechanisms are emerging. In MET addicted cells, MET inhibition leads to activation of proviral integration site for Moloney murine leukemia virus-1 (PIM1). PIM1 and proto-oncogene tyrosine-protein kinase Src (SRC) can regulate the expression of receptor tyrosine kinases (RTKs), potentially inducing resistance to MET inhibition through cross-activation. METHODS: We evaluated the activity of class I-II MET inhibitors, the SRC inhibitor dasatinib, and pan-PIM inhibitors in four MET addicted cell lines. We assessed the effect of the dual MET/PIM and MET/SRC inhibition on cell viability and at the protein level. We evaluated RNA expression profiles of the cell lines. Advanced NSCLCs were also screened for MET alterations. RESULTS: All cell lines were sensitive to class I-II MET inhibitors. All cell lines were resistant to single PIM and SRC inhibition. Dual MET/PIM inhibition was synergistic or additive in MET amplified cell lines and dual MET/SRC inhibition was highly synergistic in all MET addicted cell lines. The addition of an SRC inhibitor partially prevents the RTKs cross-activation. MET alterations were found in 9 out of 97 evaluable samples (9.3%); median overall survival in MET altered patients was 5 months (95% CI, 3 m-NA). CONCLUSIONS: We identified a potential role of PIM inhibition in MET amplified tumors and of SRC inhibition in MET addicted tumors. Potential applications of this new treatment strategy warrant further evaluation.

Serum IGF-I, IGFBP-3, and matrix metalloproteinase-7 levels and acquired chemo-resistance in advanced colorectal cancer.

Insulin-like growth factor-I (IGF-I) is thought to have antiapoptotic and mitogenic properties in colorectal cancer, whereas IGF-binding protein-3 (IGFBP-3) seems to exert a pro-apoptotic effect. Additionally, matrix metalloproteinase-7 (MMP-7), an enzyme with in vitro ability to degrade IGFBP-3, has been shown to be a prognostic factor in advanced colorectal cancer (ACRC). We studied whether chemotherapy treatment for ACRC modulates IGF-I, IGFBP-3, and MMP-7 serum levels. In 41 patients undergoing first-line therapy for ACRC, serum levels of IGF-I, IGFBP-3, and MMP-7 were measured with immunoassays at baseline and every 3 months until progressive disease, or a maximum of five determinations, during a chemotherapy regimen of either FOLFOX or FOLFIRI therapies. Associations were assessed for paired samples, using t-test or Wilcoxon ranks test depending on normality of the variable, verified with Shapiro-Wilk test. An average of four extractions (range 3-5) were done, for a total of 157 determinations. Mean pretreatment values of IGF-I, IGFBP-3, and MMP-7 were 83 (95% CI, 73-92) ng/ml, 2372 (95% CI, 2121-2623) ng/ml, and 10.6 (95% CI, 7.21-13.98) ng/ml respectively. No significant changes in IGF-I were found, but a significant increase in IGFBP-3 serum concentrations was observed during or after chemotherapy treatment without progressive disease, compared with basal levels (P<0.001). A significant decrease in IGFBP-3 to 1983 ng/ml (95% CI, 1675-2292) and a significant increase in MMP-7 levels to 14.6 (7.6-21.7) ng/ml were observed at progression of disease compared with baseline and treatment levels (P<0.001). This study shows that IGFBP-3 and MMP-7 serum levels change during chemotherapy treatment. The increased MMP-7 levels at disease progression support the hypothesis that this protease could play a role in acquired resistance by degrading IGFBP-3.

Serum matrilysin levels predict outcome in curatively resected colorectal cancer patients.

BACKGROUND: Matrix metalloproteinase 7 (MMP-7) is involved in invasion, metastasis, growth, and angiogenesis. The aim of this study is to assess the prognostic role of serum MMP-7 in curatively resected colorectal cancer (CRC). MATERIALS AND METHODS: Patients undergoing resection for CRC (n = 175) were recruited from July 2003 to December 2004. MMP-7 was determined using a quantitative solid phase sandwich ELISA. Cox analysis was used to assess the role of MMP-7 in predicting overall survival (OS) and disease-free survival (DFS). RESULTS: The median length of follow-up was 45 months (range 1 to 59). Levels of MMP-7 are predictors of DFS (hazard ratio [HR] 1.119, 95% confidence interval [95% CI] 1.038-1.207) and of OS (HR 1.113, 95% CI 1.025-1.209). Patients with MMP-7 higher than the median (4.3 ng/ml) are more likely to relapse (29.5% vs 18.4%, P = .084); median time to progression in relapsed patients is 8 months if MMP-7 is > or =4.3 ng/ml and 18 months if MMP-7 is <4.3 ng/ml. Node-negative patients with low MMP-7 have a predicted probability of relapse-free survival at 4 years of 88% (95% CI 83-92%); if the MMP-7 is higher than the median value; this probability is 77% (95% CI 73-81%). CONCLUSION: MMP-7 predicts recurrence in curatively resected CRC patients.

Osimertinib and pterostilbene in EGFR-mutation-positive non-small cell lung cancer (NSCLC).

Monotherapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) still leads to incomplete responses in most EGFR-mutation positive non-small cell lung cancer (NSCLC) patients, often due to acquired resistance through activation of parallel compensatory pathways. We have previously shown that co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3), and Src-yes-associated protein 1 (YAP1) was highly synergistic in vitro and in vivo. In the present study, we treated EGFR-mutation positive cell lines with the combination of osimertinib plus a natural compound, pterostilbene, which has been reported to abrogate Src and STAT3 activation. Methods: Cell viability assays and immunoblotting were performed to reveal the mechanisms of action of pterostilbene, osimertinib and pterostilbene plus osimertinib in five EGFR-mutation positive NSCLC and one triple negative breast cancer (TNBC) cell lines. Results: Osimertinib plus pterostilbene yielded synergistic effects in all EGFR-mutation positive NSCLC cell lines investigated. Surprisingly, pterostilbene alone did not inhibit, nor downregulate Src phosphorylation in the EGFR-mutation positive NSCLC cell lines or the TNBC cell line, MDA-MB-231. However, the double combination of osimertinib plus pterostilbene reversed the osimertinib-induced STAT3, YAP1, and CUB domain-containing protein-1 (CDCP1) phosphorylation and slightly suppressed Src phosphorylation in PC9 and H1975 cells. Conclusion: The results of this study indicate that pterostilbene may be used to abrogate the activated resistance pathways of single osimertinib treatment in EGFR-mutation positive NSCLC. Future studies should focus on in vivo translation and confirmation of these results.

Hsp90 inhibitors enhance the antitumoral effect of osimertinib in parental and osimertinib-resistant non-small cell lung cancer cell lines.

BACKGROUND: Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. METHODS: MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. RESULTS: Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. CONCLUSIONS: Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition.