Correction: Meisaprow et al. Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, ß3, and FAK/Akt/c-Myc Signaling Pathway. Molecules 2021, 26, 7659.

In the original publication [...].

Efficacy and mechanisms of cannabis oil for alleviating side effects of breast cancer chemotherapy (CBC2): protocol for randomized controlled trial.

BACKGROUND: In a pilot study using both cannabidiol (CBD) and tetrahydrocannabinol (THC) as single agents in advanced cancer patients undergoing palliative care in Thailand, the doses were generally well tolerated, and the outcome measure of total symptom distress scores showed overall symptom benefit. The current study aims to determine the intensity of the symptoms experienced by breast cancer patients, to explore the microbiome profile, cytokines, and bacterial metabolites before and after the treatment with cannabis oil or no cannabis oil, and to study the pharmacokinetics parameters and pharmacogenetics profile of the doses. METHODS: A randomized, double-blinded, placebo-controlled trial will be conducted on the breast cancer cases who were diagnosed with breast cancer and currently receiving chemotherapy at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand. Block randomization will be used to allocate the patients into three groups: Ganja Oil (THC 2 mg/ml; THC 0.08 mg/drop, and CBD 0.02 mg/drop), Metta Osot (THC 81 mg/ml; THC 3 mg/drop), and placebo oil. The Edmonton Symptom Assessment System (ESAS), Food Frequency Questionnaires (FFQ), microbiome profile, cytokines, and bacterial metabolites will be assessed before and after the interventions, along with pharmacokinetic and pharmacogenetic profile of the treatment during the intervention. TRIAL REGISTRATION: TCTR20220809001.

Impact of LKB1 status on radiation outcome in patients with stage III non-small-cell lung cancer.

Preclinical studies suggest that loss of LKB1 expression renders cancer cells less responsive to radiation partly through NRF2-mediated upregulation of antioxidant enzymes protecting against radiation-induced DNA damage. Here we investigated the association of an alteration in this pathway with radio-resistance in lung cancer patients. Patients with locally advanced non-small cell lung cancer (LA-NSCLC) who were treated with chemoradiotherapy (CRT) and analyzed for LKB1 expression using semiquantitative immunohistochemistry. Clinical characteristics and expression of LKB1 were analyzed for association with radiotherapy outcomes. We analyzed 74 available tumor specimens from 178 patients. After a median follow-up of 40.7 months, 2-year cumulative incidence of locoregional recurrence (LRR) in patients who had LKB1Low expression was significantly higher than those with LKB1High expression (68.8% vs. 31.3%, P = 0.0001). LKB1Low expression was found significantly associated with a higher incidence of distant metastases (DM) (P = 0.0008), shorter disease-free survival (DFS) (P = 0.006), and worse overall survival (OS) (P = 0.02) compared to LKB1High expression. Moreover, patients with LKB1Low expression showed a significantly higher 2-year cumulative incidence of LRR (77.6% vs. 21%; P = 0.02), higher DM recurrence (P = 0.002), and shorter OS (P < 0.0001) compared with the EGFR-mutant group. For all patients with LKB1Low who had LRR, these recurrences occurred within the field of radiation, in contrast to those with LKB1High expression having both in-field, marginal, and out-of-field failures. LKB1 expression may serve as a potential biomarker for poor outcomes after receiving radiation in LA-NSCLC patients. Further studies to confirm the association and application are warranted.

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction.

BACKGROUND: Digital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. METHODS: Study patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. RESULTS: 61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. CONCLUSION: Using ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.

A prospective randomized study comparing the efficacy between povidone-iodine gargling and benzydamine hydrochloride for mucositis prevention in head and neck cancer patients receiving concurrent chemoradiotherapy.

Background: Concurrent chemoradiation (CCRT) has been the standard treatment for organ preservation or locally advanced head and neck cancer (LAHNC). Radiation-induced oral mucositis (RIOM) is an important treatment-limiting toxicity. Benzydamine hydrochloride was recommended to prevent oral mucositis. Povidone-iodine had also been adopted to use as an oral rinse to prevent mucositis. Objective: This study compared the efficacy between benzydamine hydrochloride and 0.1% povidone-iodine to prevent RIOM in HNC patients who received concurrent chemoradiotherapy. Methods: We conducted a randomized control study in HNC patients receiving CCRT with curative intent. The stratification factors were primary site of disease, treatment modality, chemotherapy regimen, and schedule. The primary outcome was RIOM assessed by Oral Mucositis Assessment Scale (OMAS). Secondary outcomes included RIOM assessed by NCI-CTCAE, use of analgesic, antibiotics and anti-fungal drugs, hospitalization, and participant satisfaction. Results: There were 83 participants recruited for this study with 71 completing the trial. Demographic characteristics were well-balanced between both arms. The univariate regression analysis revealed that povidone-iodine correlated with less RIOM compared to benzydamine hydrochloride (coefficient -2.25, 95% CI -4.37 to -0.012, p-value 0.03). The incidence of grade III-IV CTCAE RIOM during the study period was 51.4% with benzydamine hydrochloride compared to 26.5% with 0.1% povidone iodine (p-value 0.032). The peak incidence of grade III-IV CTCAE RIOM occurred in the 7th week of treatment (40.5% vs. 11.8%, p-value 0.01). This indicated the efficacy of povidone-iodine to prevent severe RIOM which usually most severity in the last week of CCRT treatment. The multivariate analysis revealed that the CCRT setting (definitive vs. adjuvant) and gargling agents (povidone-iodine vs. benzydamine hydrochloride were the factors associated with RIOM. Conclusion: This study demonstrated higher efficacy of 0.1% povidone-iodine gargle than benzydamine hydrochloride in mucositis prevention.

Inhibition of histone deacetylase 6 destabilizes ERK phosphorylation and suppresses cancer proliferation via modulation of the tubulin acetylation-GRP78 interaction.

BACKGROUND: The leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown. METHODS: The clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT-PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells. RESULTS: HDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth. CONCLUSIONS: HDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Differential expression of immune-regulatory proteins C5AR1, CLEC4A and NLRP3 on peripheral blood mononuclear cells in early-stage non-small cell lung cancer patients.

Changes in gene expression profiling of peripheral blood mononuclear cells (PBMC) appear to represent the host's response to the cancer cells via paracrine signaling. We speculated that protein expression on circulating T-lymphocytes represent T-lymphocyte trafficking before infiltration into the tumor microenvironment. The possibility of using protein expression on circulating T-lymphocytes as a biomarker to discriminate early-stage non-small cell lung cancer (NSCLC) was explored. Four independent PBMC gene expression microarray datasets (GSE12771, GSE13255, GSE20189 and GSE3934) were analyzed. We selected C5AR1, CLEC4A and NLRP3 based on their significant protein expression in tumor-infiltrating lymphocytes, but not in normal lymphoid tissue. A validation study using automated flow cytometry was conducted in 141 study participants including 76 treatment-naive early-stage non-small cell lung cancer patients (NSCLC), 12 individuals with non-malignant pulmonary diseases, and 53 healthy individuals. Median ratios of C5AR1, CLEC4A and NLRP3 specific antibody staining to CD3 positive cells in early-stage NSCLC patients compared to healthy controls were 0.014 [0-0.37] vs. 0.01 [0-0.07, p = 0.13], 0.03 [0-0.87] vs. 0.02 [0-0.13, p = 0.10] and 0.19 [0-0.60] vs. 0.09 [0.02-0.31, p < 0.0001], respectively. Median fluorescence intensity (MFI) of CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ expression in early-stage NSCLC patients compared to healthy volunteers was 185 [64.2-4801] vs. 107.5 [27-229, p < 0.0001], 91.2 [42.4-2355] vs. 71.25 [46.2-103, p = 0.0005], and 1585 [478-5224] vs. 758.5 [318-1976, p < 0.0001], respectively. NLRP3:CD3 ratio, CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ MFI were significantly higher in early-stage NSCLC than healthy volunteers with an area under the ROC curve of 0.69-0.76. The CD3+NLRP3+ MFI provided the most distinguishable expression at 71.5% sensitivity and 70% specificity. Furthermore, CD3+NLRP3+ MFI potentially discriminated between early-stage NSCLC from malignant-mimic inflammation and infection pulmonary disease. Further validation in various pulmonary inflammatory disease might be warranted. Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes.

Prognostic factors in completely resected lymph-node-negative pulmonary adenocarcinoma.

Background: Lymph node involvement is one of the important prognostic factors for early-stage lung cancer. However, in lymph node-negative (N0) lung cancer the recurrent rate may be as high as 30%. We aimed to study potential prognostic factors including clinicopathological factors and epidermal growth factor receptor (EGFR) mutation status in this lung cancer population. Methods: We retrospectively reviewed the medical records and pathological examinations of patients with completely resected N0 pulmonary adenocarcinoma treated in our institute between 2009 and 2016. We used Cobas® test to determine EGFR mutation status. Recurrence-free survival (RFS) was analyzed by univariable and multivariable Cox regression analyses. Results: We recruited 220 patients with median duration of follow up 5 years. Majority of these patients were in stage I (80%) and did not receive adjuvant therapy (86%). There were 53% with EGFR mutations which comprised of exon 19 deletion 51% and L858R 43%. Recurrence occurred in 64 out of 220 patients (29%). The median time to recurrence was 2.1 years. Statistically significant prognostic factors in both univariate and multivariate analyses included tumor size ≥4 centimeter (cm) (HR: 1.94; 95% CI: 1.03-3.67), visceral pleural invasion (HR: 2.53; 95% CI: 1.34-4.79), tumor necrosis (HR: 2.45; 95% CI: 1.13-5.31) and bronchial resection margin <2 cm (HR: 1.96; 95% CI: 1.10-3.51). However, presence of sensitizing EGFR mutation was not found to be a significant prognostic factor (HR: 1.20; 95% CI: 0.66-2.18; P=0.56). Conclusions: In N0 surgically resected lung adenocarcinoma, there were significant pathological prognostic factors including tumor 4 cm or more, visceral pleural invasion, tumor necrosis and bronchial resection margin less than 2 cm. Mutation of EGFR is not a significant prognostic factor to determine the risk of recurrence in this population and their risks shall be determined by the other poor prognostic factors.

Aspiletrein A Induces Apoptosis Cell Death via Increasing Reactive Oxygen Species Generation and AMPK Activation in Non-Small-Cell Lung Cancer Cells.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.

Co-occurrence CDK4/6 amplification serves as biomarkers of de novo EGFR TKI resistance in sensitizing EGFR mutation non-small cell lung cancer.

Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) remains an issue of concern. We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 years). Uncommon EGFR mutation subtypes were significantly variable enriched in de novo resistance. The remaining sensitizing EGFR mutation subtypes (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-ß, p53 and ß-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6 × 10-4. We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36-73%] compared to IRs/LTRs [range 4-22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4/6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80-7.31, p-value < 0.001]. The presence of CDK4/6 amplification in pretreatment specimen serves as a predictive biomarker for de novo resistance in sensitizing EGFR mutation.

Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, ß3, and FAK/Akt/c-Myc Signaling Pathway.

Lung cancer is recognized as a major cause of mortality worldwide owing to its metastatic activity. Given the lack of solid information regarding the possible effects of caffeine, one of the most consumed natural psychoactive substances, on molecular signaling pathways implicated in the aggressive behavior of lung cancer, our study aimed to evaluate the effect and mechanism of caffeine on metastasis-related mechanisms. The results revealed that caffeine treatment at concentrations of 0-500 µM caused no direct cytotoxic effects on NCI-H23 cells. Treatment of cells with caffeine showed good potential to inhibit cell proliferation at 48 h and induced significant cell cycle arrest at the G0/G1 phase. Concerning metastasis, caffeine was shown to reduce filopodia formation, inhibit migration and invasion capability, and reduce the ability of cancer cells to survive and grow in an anchorage-independent manner. Moreover, caffeine could attenuate the formation of 3D tumor spheroids in cancer stem cell (CSC)-enriched populations. With regard to mechanisms, we found that caffeine significantly altered the integrin pattern of the treated cells and caused the downregulation of metastasis-associated integrins, namely, integrins αv and ß3. Subsequently, the downstream signals, including protein signaling and transcription factors, namely, phosphorylated focal adhesion kinase (p-FAK), phosphorylated protein kinase B (p-Akt), cell division cycle 42 (Cdc42), and c-Myc, were significantly decreased in caffeine-exposed cells. Taken together, our novel data on caffeine-inhibiting mechanism in relation to metastasis in lung cancer could provide insights into the impact of caffeine intake on human diseases and conditions.

GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy.

Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

Pongol Methyl Ether Inhibits Akt and Suppresses Cancer Stem Cell Phenotypes in Lung Cancer Cells.

Cancer stem cells (CSCs) are an important therapeutic target. The therapeutic agents targeting CSCs should lead to improved clinical outcomes. Here we have demonstrated the CSC-suppressing activity of pongol methyl ether (PME), a pure compound from Millettia erythrocalyx. METHODS: CSC-suppressing effects were evaluated by spheroid formation assay and detection of CSC markers. The related CSC cell signals were evaluated by Western blot, immunofluorescence and molecular docking analysis. Proteins affected by PME treatment were subjected to bioinformatic analysis. Protein-protein interaction (PPI) networks were constructed by the Search Tool for Interactions of Chemicals (STITCH). The Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper were used to confirm the underlying pathways. RESULTS: PME (5-25 µM) significantly suppressed the ability of lung cancer cells to form colonies, grow in an anchorage-independent manner and generate tumour spheroids. PME at 25 µM significantly decreased the CSC markers (CD133 and ALDH1A1) and pluripotent transcription factors (Oct4 and Nanog). Akt, the key upstream signal of CSC control, was significantly decreased by the PME treatment. The molecular docking indicated that PME was bound to Akt-1 with a binding affinity of -9.2 kcal/mol greater than the Akt-1 inhibitor (reference compound; CQW). The STITCH network identified a total of 15 proteins interacted in PPI networks, and Akt-1 was identified as a central protein. The KEGG mapper indicated that the selected CSC markers were mostly involved in the 'signalling pathways regulating pluripotency of stem cells' pathway map and Akt, Oct4 and Nanog were the regulatory proteins in the dominant pathway. In addition, PME (10-25 µM) can suppress spheroid formation and reduce CSC-specific marker expression in patient-derived primary lung cancer cells. CONCLUSIONS: Our study revealed a novel pharmacological effect and the underlying mechanism of PME that can attenuate CSC phenotypes in lung cancer cells and may be developed for lung cancer therapy.

Titania Nanosheet Generates Peroxynitrite-Dependent S-Nitrosylation and Enhances p53 Function in Lung Cancer Cells.

Metal nanomaterials can enhance the efficacy of current cancer therapies. Here, we show that Ti0.8O2 nanosheets cause cytotoxicity in several lung cancer cells but not in normal cells. The nanosheet-treated cells showed certain apoptosis characteristics. Protein analysis further indicated the activation of the p53-dependent death mechanism. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses revealed the cellular uptake of the nanosheets and the induction of cell morphological change. The nanosheets also exhibited a substantial apoptosis effect on drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than standard drugs. Ti0.8O2 nanosheets induce apoptosis through a molecular mechanism involving peroxynitrite (ONOO-) generation. As peroxynitrite is known to be a potent inducer of S-nitrosylation, we further found that the nanosheets mediated the S-nitrosylation of p53 at C182, resulting in higher protein-protein complex stability, and this was likely to induce the surrounding residues, located in the interface region, to bind more strongly to each other. Molecular dynamics analysis revealed that S-nitrosylation stabilized the p53 dimer with a ΔGbindresidue of <-1.5 kcal/mol. These results provide novel insight on the apoptosis induction effect of the nanosheets via a molecular mechanism involving S-nitrosylation of the p53 protein, emphasizing the mechanism of action of nanomaterials for cancer therapy.

Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRß, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M-1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.

Efficacy of Combination Docetaxel and Nintedanib in Advanced Non-Small Cell Lung Cancer in Thailand: A Multicenter Study.

INTRODUCTION: The mainstay systemic treatment for non-oncogenic addictive advanced stage non-small cell lung cancer is chemotherapy. Anti-angiogenic agents are additive compounds that enhance disease control and lead to improvement of overall survival benefit. Recently PD-(L)1 blockage, a checkpoint inhibitor, has been adopted as another line of treatment. A sequential strategy to enhance the efficacy of combination docetaxel and nintedanib after immunotherapy, correlated with genomic mutation, has been explored. METHOD: A retrospective cohort study of 56 patients from 8 centers in Thailand who received combination docetaxel and nintedanib via the Thai nintedanib Named Patient Use program was conducted. Demographic characteristics, treatment details, and treatment responses were retrieved from medical records. RESULTS: The majority of patients were male (62.5%) with adenocarcinoma subtype (88%). Thirty-five percent had sensitizing EGFR mutation. Combination docetaxel and nintedanib was given as second to fourth line of treatment. Median PFS of docetaxel/nintedanib was 5.6 months [95% CI 4.8-6.9]. Median OS of the entire cohort was 22.5 months [95% CI 20.2-31.1]. Among them, only four patients received this combination after immunotherapy which limited the validity of efficacy analysis. Median PFS of those four patients was 7.9 months [range 5.2-9.1] which was slightly higher than the remaining cohort (median PFS 4.5 months, 95% CI: 4.0-6.0, p-value 0.09). Among the adenocarcinoma subtype, a relapse-time of platinum-doublet chemotherapy of more than 6 months was solely indicated as a benefit of combination docetaxel/nintedanib treatment compared to the relapse-time of platinum-doublet chemotherapy of less than 6 months by multivariate HR of PFS 0.32 [95% CI: 0.14-0.68, p-value 0.003]. CONCLUSION: Combination docetaxel and nintedanib provided more benefit in relapse-time of platinum-doublet chemotherapy of more than 6 months in advanced stage adenocarcinoma lung cancer. Neither EGFR nor ALK alteration influenced the outcome of treatment.

Artocarpin Targets Focal Adhesion Kinase-Dependent Epithelial to Mesenchymal Transition and Suppresses Migratory-Associated Integrins in Lung Cancer Cells.

Focal adhesion kinase (FAK) controls several cancer aggressive potentials of cell movement and dissemination. As epithelial-mesenchymal transition (EMT) and the migratory-associated integrins, known influencers of metastasis, have been found to be linked with FAK activity, this study unraveled the potential pharmacological effect of artocarpin in targeting FAK resulting in the suppression of EMT and migratory behaviors of lung cancer cells. Treatment with artocarpin was applied at concentrations of 0-10 µM, and the results showed non-cytotoxicity in lung cancer cell lines (A549 and H460), normal lung (BEAS-2B) cells and primary metastatic lung cancer cells (ELC12, ELC16, and ELC20). We also found that artocarpin (0-10 µM) had no effect on cell viability, proliferation, and migration in BEAS-2B cells. For metastasis-related approaches, artocarpin significantly inhibited cell migration, invasion, and filopodia formation. Artocarpin also dramatically suppressed anchorage-independent growth, cancer stem cell (CSC) spheroid formation, and viability of CSC-rich spheroids. For molecular targets of artocarpin action, computational molecular docking revealed that artocarpin had the best binding affinity of -8.0 kcal/mol with FAK protein. Consistently, FAK-downstream proteins, namely active Akt (phosphorylated Akt), active mTOR (phosphorylated mTOR), and Cdc42, and EMT marker and transcription factor (N-cadherin, Vimentin, and Slug), were found to be significantly depleted in response to artocarpin treatment. Furthermore, we found the decrease of Caveolin-1 (Cav-1) accompanied by the reduction of integrin-αν and integrin-ß3. Taken together, these findings support the anti-metastasis potentials of the compound to be further developed for cancer therapy.

Tubulin acetylation enhances lung cancer resistance to paclitaxel-induced cell death through Mcl-1 stabilization.

The posttranslational modifications (PTMs) of microtubules have been reported to play an important role in cancer aggressiveness, including apoptosis resistance. In this study, we aimed to investigate the biological role of microtubule PTMs in the regulation of paclitaxel responsiveness. The acetylated tubulin (Ace-tub) level was strongly associated with paclitaxel sensitivity, as observed in patient-derived primary lung cancer cells and xenografted immunodeficient mice. We showed that paclitaxel-resistant H460 lung cancer cells, generated by a stepwise increase in paclitaxel, exhibited markedly increased tubulin acetylation and consequently acquired paclitaxel resistance. Upregulation of tubulin acetylation by overexpression of α-tubulin acetyltransferase 1 wild-type (αTAT1wt), an enzyme required for acetylation, or by treatment with trichostatin A (TSA), a histone deacetylase 6 (HDAC6) inhibitor, significantly attenuated paclitaxel-induced apoptosis. Investigation of the underlying mechanism revealed that the levels of antiapoptotic Mcl-1 appeared to increase in αTAT1wt-overexpressing and TSA-treated cells compared to control cells, whereas the levels of other antiapoptotic regulatory proteins were unchanged. On the other hand, decreased tubulin acetylation by αTAT1 RNA interference downregulated Mcl-1 expression in patient-derived primary lung cancer and paclitaxel-resistant lung cancer cells. A microtubule sedimentation assay demonstrated that Mcl-1 binds to microtubules preferentially at Ace-type, which prolongs the Mcl-1 half-life (T1/2). Furthermore, immunoprecipitation analysis revealed that polyubiquitination of Mcl-1 was extensively decreased in response to TSA treatment. These data indicate that tubulin acetylation enhances the resistance to paclitaxel-induced cell death by stabilizing Mcl-1 and protecting it from ubiquitin-proteasome-mediated degradation.

Bibenzyl analogue DS-1 inhibits MDM2-mediated p53 degradation and sensitizes apoptosis in lung cancer cells.

BACKGROUND: Lung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function. PURPOSE: The present study aimed to investigate the effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells. METHODS: The efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed. RESULTS: DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon-hydrogen bond interaction at Lys27, π-alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity. CONCLUSIONS: Our findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.

Vimentin expression status is a potential biomarker for brain metastasis development in EGFR-mutant NSCLC patients.

BACKGROUND: Despite advances in systemic therapy and improvements in survival for advanced epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC), brain metastasis (BM) remains a poor outcome. Previous studies on risk factors for BM occurrence included unselected patients and biomarker prediction of BM in these populations were not well studied. We aimed to identify the role of epithelial mesenchymal transition (EMT) marker and clinical factors predicting BM in. EGFR: mutant NSCLC patients. METHODS: Advanced EGFR-mutant NSCLC patients in the King Chulalongkorn Memorial Hospital from January 2013 to December 2017 were included. Vimentin expression was assessed by immunohistochemistry. The correlation between vimentin expression and factors associated with BM occurrence was analyzed by univariate and multivariate analyses. RESULTS: 304 patients were enrolled. Of these, 149 patients (49%) developed BM. In multivariate analysis, the occurrence of BM was associated with age <60 years, metastatic disease at diagnosis, and 3 or more metastatic sites. Moreover, positive vimentin expression was also found more common in patients with BM than those without BM (52.4% vs. 27.6%, respectively) and predicted overall BM development in EGFR-mutant patients (OR 2.53, 95% CI, 1.11-5.77; P=0.027). Overall survival (OS) was shorter in vimentinpositive group than in vimentinnegative group. Median OS was 20.0 months (95% CI, 14.51-25.51) and 30.9 months (95% CI, 20.99-40.84), respectively (HR, 1.57; P=0.04). CONCLUSIONS: Younger patients with EGFR-mutant NSCLC who had high disease burden were more likely to develop BM. Vimentin served as a biomarker for predicting BM and poor prognostic factor in EGFR-mutant patients. EMT pathway may be considered as a therapeutic target in these high-risk populations.