Erlotinib Treatment Research Articles

Precision-cut liver slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis.

Considering the lack of successful treatment options and poor prognosis for cirrhosis and cirrhosis-induced HCC, new platforms to investigate antifibrotic therapies are urgently needed. Precision-cut liver slice (PCLS) is a powerful ex vivo culture model that can supplement and potentially replace the traditional models. PCLS were prepared from 4 different murine cirrhotic models (choline-deficient, l-amino acid-defined, high-fat diet, thioacetamide, diethylnitrosamine, and carbon tetrachloride) and compared with in vivo murine experiments, in vitro hepatic stellate cells, and human cirrhotic PCLS. PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGF receptor inhibitor, significantly inhibited profibrogenic gene expressions in PCLS from choline-deficient, l-amino acid-defined, high-fat diet or thioacetamide-induced cirrhotic rats. Erlotinib treatment of PCLS from diethylnitrosamine or carbon tetrachloride-induced cirrhotic rats inhibited the expression of profibrogenic genes, which was consistent with the impact of erlotinib on these genes in in vivo diethylnitrosamine or carbon tetrachloride-induced cirrhosis. In addition, in hepatic stellate cells at PCLS from normal mice, erlotinib treatment inhibited TGF-β1-upregulated expression of Acta2. Similar expression results were observed in in vitro hepatic stellate cells. Expression of key regulators of fibrosis progression and regression were also significantly altered. Changes in profibrogenic gene expression under erlotinib treatment were also corroborated with human cirrhotic PCLS. Responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. These results were verified in human cirrhotic PCLS. PCLS is an excellent model for assessing antifibrotic therapies that are aligned with the principles of replacement, reduction, and refinement (3Rs), and it will benefit preclinical and clinical research for human fibrosis and cirrhosis.

Erlotinib regulates short-term memory, tau/Aβ pathology, and astrogliosis in mouse models of AD.

Erlotinib is an epidermal growth factor receptor (EGFR) inhibitor that is approved by the FDA to treat non-small cell lung cancer (NSCLC). Several membrane receptors, including EGFR, interact with amyloid β (Aβ), raising the possibility that erlotinib could have therapeutic effects on Alzheimer's disease (AD). However, the effects of erlotinib on Aβ/tau-related pathology and cognitive function in mouse models of AD and its mechanisms of action have not been examined in detail. To investigate the effects of erlotinib on cognitive function and AD pathology, 3 to 6-month-old PS19 mice and 3 to 3.5-month-old 5xFAD mice and WT mice were injected with vehicle (5% DMSO + 10% PEG + 20% Tween80 + 65% D.W.) or erlotinib (20 mg/kg, i.p.) daily for 14 or 21 days. Then, behavioral tests, Golgi staining, immunofluorescence staining, western blotting ELISA, and real-time PCR were conducted. We found that erlotinib significantly enhanced short-term spatial memory and dendritic spine formation in 6-month-old P301S tau transgenic (PS19) mice. Importantly, erlotinib administration reduced tau phosphorylation at Ser202/Thr205 (AT8) and Thr231 (AT180) and further aggregation of tau into paired helical fragments (PHFs) and neurofibrillary tangles (NFTs) in 3-month-old and/or 6-month-old PS19 mice by suppressing the expression of the tau kinase DYRK1A. Moreover, erlotinib treatment decreased astrogliosis in 6-month-old PS19 mice and reduced proinflammatory responses in primary astrocytes (PACs) from PS19 mice. In 3- to 3.5-month-old 5xFAD mice, erlotinib treatment improved short-term spatial memory and hippocampal dendritic spine number and diminished Aβ plaque deposition and tau hyperphosphorylation. Furthermore, erlotinib-treated 5xFAD mice exhibited significant downregulation of astrocyte activation, and treating PACs from 5xFAD mice with erlotinib markedly reduced cxcl10 (reactive astrocyte marker) and gbp2 (A1 astrocyte marker) mRNA levels and proinflammatory cytokine mRNA and protein levels. Taken together, our results suggest that erlotinib regulates tau/Aβ-induced AD pathology, cognitive function, and Aβ/tau-evoked astrogliosis and therefore could be a potent therapeutic drug for ameliorating AD symptoms.

Erlotinib Treatment in Colorectal Cancer Suppresses Autophagy Based on KRAS Mutation.

The KRAS gene is mutated in approximately 45% of colorectal cancer patients. There are currently very few targeted treatments or therapies equipped to directly inhibit KRAS due to its unusual structural intricacies. Erlotinib, an EGFR inhibitor, has previously been demonstrated to reduce cell viability by inducing autophagy in lung cancer cell lines with varying EGFR mutations. In contrast to lung cancer cells, evidence is provided herein for the first time that erlotinib treatment in colorectal cancer (CRC) cell lines reduces autophagy and still results in decreased cell viability. However, the effects of erlotinib in CRC cell lines containing a wildtype KRAS gene were different than in cells carrying a mutant KRAS gene. We show that there is significantly more downregulation of autophagy in KRAS mutant CRC cells compared to KRAS wildtype cells, both at transcriptional and translational levels, suggesting that the KRAS mutation is advantageous for cancer growth, even in the presence of erlotinib. Cell viability results determined that KRAS wildtype CRC cells had significantly more cell death compared to KRAS mutant cells. Using patient mRNA datasets, we showed that there was a significant correlation between the presence of the KRAS mutation and the expression of autophagy proteins. Additionally, through molecular dynamics simulations, we develop a blueprint for KRAS and autophagy protein interaction and the impact of the KRAS mutation on autophagy protein regulation. Overall, this is the first report of erlotinib treatment in CRC cells that assesses autophagy, and we demonstrate that autophagy activity is downregulated in these cells. This effect is not only greater in cells carrying a KRAS mutation compared to wildtype cells, but the KRAS mutant cells also have increased cell viability compared to wildtype cells. We hypothesize that the difference in cell viability and autophagy expression between KRAS mutant and KRAS wildtype cells after treatment with erlotinib can be of therapeutic value to treat CRC patients carrying KRAS mutations.

Combination of monensin and erlotinib synergistically inhibited the growth and cancer stem cell properties of triple-negative breast cancer by simultaneously inhibiting EGFR and PI3K signaling pathways.

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are recognized as a highly challenging subset of cells, renowned for their heightened propensity for relapse and unfavorable prognosis. Monensin, an ionophoric antibiotic, has been reported to exhibit significant therapeutic efficacy against various cancers, especially CSCs. Erlotinib is classified as one of the EGFR-TKIs and has been previously identified as a promising therapeutic target for TNBC. Our research aims to assess the effectiveness of combination of monensin and erlotinib as a potential treatment strategy for TNBC. The combination of monensin and erlotinib was assessed for its potential anticancer activity through various in vitro assays, including cytotoxicity assay, colony formation assay, wound healing assay, transwell assay, mammosphere formation assay, and proportion of CSCs assay. Additionally, an in vivo study using tumor-bearing nude mice was conducted to evaluate the inhibitory effect of the monensin and erlotinib combination on tumor growth. The results indicated that combination of monensin with erlotinib synergistically inhibited cell proliferation, the migration rate, the invasion ability and decreased the CSCs proportion, and CSC markers SOX2 and CD133 in vivo and in vitro. Furthermore, the primary proteins involved in the signaling pathways of the EGFR/ERK and PI3K/AKT are simultaneously inhibited by the combination treatment of monensin and erlotinib in vivo and in vitro. The simultaneous inhibition of the EGFR/ERK and PI3K/AKT/mTOR signaling pathways by the combination of monensin and erlotinib exhibited a synergistic effect on suppressing tumor proliferation and cancer cell stemness in TNBC.

Multidrug-Resistant Profiles in Non-Small Cell Lung Carcinoma Patient-Derived Cells: Implications for Personalized Approaches with Tyrosine Kinase Inhibitors.

The impact of tyrosine kinase inhibitors (TKIs) on multidrug resistance (MDR) in non-small cell lung carcinoma (NSCLC) is a critical aspect of cancer therapy. While TKIs effectively target specific signaling pathways of cancer cells, they can also act as substrates for ABC transporters, potentially triggering MDR. The aim of our study was to evaluate the response of 17 patient-derived NSCLC cultures to 10 commonly prescribed TKIs and to correlate these responses with patient mutational profiles. Using an ex vivo immunofluorescence assay, we analyzed the expression of the MDR markers ABCB1, ABCC1, and ABCG2, and correlated these data with the genetic profiles of patients for a functional diagnostic approach. NSCLC cultures responded differently to TKIs, with erlotinib showing good efficacy regardless of mutation burden or EGFR status. However, the modulation of MDR mechanisms by erlotinib, such as increased ABCG2 expression, highlights the challenges associated with erlotinib treatment. Other TKIs showed limited efficacy, highlighting the variability of response in NSCLC. Genetic alterations in signaling pathways associated with drug resistance and sensitivity, including TP53 mutations, likely contributed to the variable responses to TKIs. The relationships between ABC transporter expression, gene alterations, and response to TKIs did not show consistent patterns. Our results suggest that in addition to mutational status, performing functional sensitivity screening is critical for identifying appropriate treatment strategies with TKIs. These results underscore the importance of considering drug sensitivity, off-target effects, MDR risks, and patient-specific genetic profiles when optimizing NSCLC treatment and highlight the potential for personalized approaches, especially in early stages.

Real-world osimertinib pretreatment experience in patients with epidermal growth factor receptor T790M mutation-positive locally advanced or metastatic non-small cell lung cancer.

Osimertinib has demonstrated efficacy in patients with epidermal growth factor receptor (EGFR) T790M-positive non-small cell lung cancer (NSCLC) in clinical trials. However, real-world data on its effectiveness remain scarce. Taiwanese patients with T790M-positive locally advanced or metastatic NSCLC and progressive disease following treatment with at least one EGFR tyrosine kinase inhibitor (TKI) were enrolled from the osimertinib early access program. Of the 419 patients (mean age, 63 years; female, 67%), 53% were heavily pretreated (≥ third-line [3L]), making osimertinib a fourth-line (4L) intervention. The median progression-free survival (PFS) was 10.5 months (95% confidence interval [CI]: 8.95-11.41); the 18-month PFS rate was 26.5%. The median overall survival (OS) was 19.0 months (95% CI: 16.30-20.95); the 24-month OS rate was 40.9%. The objective response rate was 32.46%, and the disease control rate was 86.38%. The median time to treatment discontinuation of osimertinib monotherapy was 11.9 months (95% CI: 10.49-13.11). Subgroup analyses of median PFS and OS in the chemotherapy combination group vs. the osimertinib monotherapy group yielded no difference. Central nervous system (CNS) metastasis, number of prior lines of therapy, and types of initial EGFR-TKIs did not significantly impact outcomes. The median PFS values were 9.0 (95% CI: 5.18-11.34) and 10.9 (95% CI: 9.18-11.90) months with and without CNS metastasis, respectively, and 10.8 (95% CI: 8.59-12.69), 13.6 (95% CI: 10.89-16.3), and 9.2 (95% CI: 7.8-10.62) months for second-line (2L), 3L, and ≥4L therapy, respectively. In patients who received osimertinib as 2L therapy, the median PFS values in response to prior afatinib, erlotinib and gefitinib treatment were 11.2 (95% CI: 4.85-4.79), 10.5 (95% CI: 8.59-20.26) and 8.7 (95% CI: 7.21-16.79) months, respectively. Overall, real-world data from Taiwan support the clinical benefits of osimertinib in EGFR T790M -positive NSCLC.

Evaluation of EGFR and COX pathway inhibition in human colon organoids of serrated polyposis and other hereditary cancer syndromes.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Abstract 5162: Tumor transcriptional identity as a key predictor of clinical outcome in EGFR-Mutant non-small cell lung cancer

Abstract Background: EGFR tyrosine-kinase inhibitors (TKIs) produce significant clinical benefit in patients (pts) harboring EGFR-activating mutations, however, patient outcomes remain heterogeneous, highlighting the need to identify novel biomarkers for treatment response prediction. We aim to investigate the association of tumor transcriptional identity with clinical outcomes in EGFR-mutant NSCLC. Methods: We used consensus classification to derive unsupervised transcriptionally distinct clusters in EGFR-mutant NSCLC from the TCGA (N= 44). Then, we built a predictive model using extreme-gradient boosting to (1) classify an additional 88 surgically resected tumors from the LCCS study and analyzed for overall survival (OS) prediction; (2) classify metastatic EGFR-mutant NSCLC from the RELAY trial with available RNAseq data (N = 106) and analyzed for progression-free survival (PFS) with erlotinib treatment. Results: Consensus clustering revealed two distinct clusters, one associated with more lineage-preserving features and one with more lineage-independent features that could transcriptionally distinguish EGFR-mutant lung cancer. No differences in primary EGFR mutations were detected across the two clusters. Ingenuity Pathway Analysis revealed TGFB1 as the most significantly enriched pathway in the lineage-independent cluster which was also significantly more mesenchymal (p = 0.0028). For surgically resectioned EGFR-mutant NSCLC, OS was consistently shorter in the lineage-independent cluster for TCGA (23.6 vs 58.3 mo; p = 0.049; HR = 0.4) and the LCCS cohort (55.7 vs not-reached; p = 0.0073; HR = 0.27). In the metastatic setting with erlotinib treatment, there was a trend for PFS to be shorter in the lineage-independent cluster (15.2 vs 19.4 mo; p = 0.12; HR = 0.61). Conclusions: Transcriptional identity in EGFR-mutant lung cancer are prognostic of clinical outcome. The lineage-independent tumors have inferior benefit to EGFR TKI as well as inferior OS, indicating that transcriptional biomarkers can be independent of oncogene biomarkers. These transcriptional biomarkers may help identify pts with poor outcome for future intensification of therapies. Citation Format: Simon Heeke, Monique Nilsson, Don Gibbons, Jianjun Zhang, Ignatio I. Wistuba, Keunchil Park, John V. Heymach, Xiuning Le. Tumor transcriptional identity as a key predictor of clinical outcome in EGFR-Mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5162.

Abstract 4328: Erlotinib suppresses autophagy in colorectal cancer

Abstract Introduction: Oncogenic KRAS mutation is prevalent in approximately 45% of colorectal cancer (CRC) patients. Limited targeted therapies are available to inhibit the detrimental effects of KRAS mutation primarily pertaining to its unique structural intricacies. Erlotinib is an EGFR inhibitor which inducing autophagy in lung cancer cells. In this study, we followed the effects of Erlotinib treatment in mutant and wild-type colorectal cancer (CRC) cells. We report that Erlotinib exhibits selectivity between mutated KRAS and wild type, significantly modulating the expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A. Methodology: The 2 cell lines used in this study were HCT116 (KRAS mutant) and Hke3 (KRAS wild type). These cells were treated with Erlotinib for 24 hours and 48 hours, A cell viability assay was performed and subsequently followed by protein extraction. The expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A was determined. The TCGA COAD-READ patient dataset was utilized using GEPIA 2 software. A correlation was found between the autophagy proteins and the KRAS gene expression. Results: The cell viability assays showed that there was significantly more cell death in KRAS wild-type cells compared with KRAS mutant at time points ranging from 24 to 48 hours (p = 0.036). The experiment also showed that treatment of Erlotinib with concentrations of 20 uM, 35 uM, and 50 uM and significant down regulatory fold changes (p < 0.05). Both KRAS mutated and wild-type cells treated with Erlotinib expressed significant downregulation of ATG5, ULK1, Beclin1, and LC3A proteins at 48 hours (p < 0.05). The 20 uM treatment demonstrated a significance of p < 0.05 for all proteins. The 20 uM treatment resulted in fold changes of the KRAS mutated cells for ATG5, ULK1, Beclin1, and LC3A to be 1.42, 1.42, 1.81, and 2.09 respectively. A strong KRAS correlation was found with ATG5, ULK1, Beclin1, and LC3A using COAD & READ tumor and COAD & READ normal (R > 0.75, p < 0.05). Conclusion: This study highlights the effects of Erlotinib as a treatment for KRAS mutated CRC. Our results confirmed that Erlotinib had the opposite effect on CRC cells in relation to the expression of the autophagy proteins when compared to lung cancer cells. There were distinct effects of Erlotinib between KRAS wild-type and mutant cells in both cell viability and the expression of autophagy-related proteins. In both KRAS cell types, there was significant downregulation of autophagy proteins, however, the KRAS wild type had significantly more downregulation than the KRAS mutant. This data suggests that there is a relationship between KRAS mutation and the expression of the autophagy proteins. Our analysis of the patient datasets confirmed the strong correlation between KRAS mutation and the expression of autophagy proteins. These findings emphasize the need to explore further how Erlotinib can be augmented into the current treatment regimen as a benefit to CRC patients. Citation Format: Alexander Siegman, Aaron Shaykevich, Isaac Silverman, Sanjay Goel, Radhashree Maitra. Erlotinib suppresses autophagy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4328.

Abstract 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3290.

Activation of the PI3K/AKT signaling pathway by ARNTL2 enhances cellular glycolysis and sensitizes pancreatic adenocarcinoma to erlotinib

BackgroundPancreatic adenocarcinoma (PC) is an aggressive malignancy with limited treatment options. The poor prognosis primarily stems from late-stage diagnosis and when the disease has become therapeutically challenging. There is an urgent need to identify specific biomarkers for cancer subtyping and early detection to enhance both morbidity and mortality outcomes. The addition of the EGFR tyrosine kinase inhibitor (TKI), erlotinib, to gemcitabine chemotherapy for the first-line treatment of patients with advanced pancreatic cancer slightly improved outcomes. However, restricted clinical benefits may be linked to the absence of well-characterized criteria for stratification and dependable biomarkers for the prediction of treatment effectiveness.Methods and resultsWe examined the levels of various cancer hallmarks and identified glycolysis as the primary risk factor for overall survival in PC. Subsequently, we developed a glycolysis-related score (GRS) model to accurately distinguish PC patients with high GRS. Through in silico screening of 4398 compounds, we discovered that erlotinib had the strongest therapeutic benefits for high-GRS PC patients. Furthermore, we identified ARNTL2 as a novel prognostic biomarker and a predictive factor for erlotinib treatment responsiveness in patients with PC. Inhibition of ARNTL2 expression reduced the therapeutic efficacy, whereas increased expression of ARNTL2 improved PC cell sensitivity to erlotinib. Validation in vivo using patient-derived xenografts (PDX-PC) with varying ARNTL2 expression levels demonstrated that erlotinib monotherapy effectively halted tumor progression in PDX-PC models with high ARNTL2 expression. In contrast, PDX-PC models lacking ARNTL2 did not respond favorably to erlotinib treatment. Mechanistically, we demonstrated that the ARNTL2/E2F1 axis-mediated cellular glycolysis sensitizes PC cells to erlotinib treatment by activating the PI3K/AKT signaling pathway.ConclusionsOur investigations have identified ARNTL2 as a novel prognostic biomarker and predictive indicator of sensitivity. These results will help to identify erlotinib-responsive cases of PC and improve treatment outcomes. These findings contribute to the advancement of precision oncology, enabling more accurate and targeted therapeutic interventions.

Cuproptosis/OXPHOS tendency prediction of prognosis and immune microenvironment of esophageal squamous cell carcinoma: Bioinformatics analysis and experimental validation

BackgroundCuproptosis is a newly discovered cell death mechanism that relies on mitochondrial respiration, for which oxidative phosphorylation (OXPHOS) is an essential part. However, the detailed mechanisms of cuproptosis associated with OXPHOS in esophageal squamous cell carcinoma (ESCC) and how this correlation affects prognosis still remains unclear. MethodsscRNA-seq data of ESCC were downloaded from SRA (Sequence Read Archive) database. “AUCell” algorithm was used to grouping epithelial cells according to cuproptosis and OXPHOS score. Cell-cell communication, Pseudo-time Trajectory and transcription factor enrichment analysis were repectively conducted by “CellChat”, “monocle3” package and “pySCENIC” algorithm. Univariate and LASSO cox regression analysis were used to construct the prognostic cuproptosis-OXPHOS signature. Finally, CCK-8 assay and DCFH-DA staining assay were respectively validated the sensitive and ROS production of elesclomol. ResultsscRNA-seq data were analyzed to identify 10 core cell types. According to the median scores for cuproptosis and OXPHOS, malignant epithelial cells were divided into double high, double low, and mixed groups. The double high group distributed at the end of the pseudo-time trajectory and harbored HMGA1(+) as specific transcriptional regulons. Knockdown of HMGA1 partly reversed the inhibition of cell viability visualized by CCK-8 assay, while reactive oxygen species (ROS) production by elesclomol was enhanced after HMGA1 silencing. Furthermore, the immunosuppressive signal was significantly increased in the double high group detected by ‘CellChat’ in single-cell data and ‘ssGSEA’ in bulk data followed by ‘CIBERSORTx’ algorithm. Finally, a new cuproptosis-OXPHOS prognostic signature (CNN2, ATP6V0E1, PSMD6, CCDC25, IGFBP2, MT1E, and RPS4Y1) was constructed for the prediction of the prognosis, and a high-risk group corresponding to a more sensitive tendency to erlotinib, dasatinib, and bosutinib treatment was identified. ConclusionsOur study revealed the relationship between OXPHOS and tendency of cuproptosis in ESCC, and malignant cells with this characteristic exerted immunosuppressive signals and indicated poor prognosis. Furthermore, we constructed the regulatory network in high cuproptosis-OXPHOS ESCC and identified HMGA1 as a potential regulator molecule of cuproptosis mediated by elesclomol.

Thoracic radiation in combination with erlotinib-results from a phase 2 randomized trial.

Radiotherapy (RT) can be used to reduce symptoms and maintain open airways for patients with non-small cell lung cancer when systemic treatment is not sufficient. For some patients, tumor control is not achieved due to radioresistance. Concurrent inhibition of epidermal growth factor receptors has been proposed as a strategy to overcome radioresistance but may increase toxicity. We performed a randomized trial to assess the efficacy, tolerance, and quality of life of concurrent erlotinib and palliative thoracic RT for patients with advanced non-small cell lung cancer. Patients were randomized 1:1 to RT alone (arm A) or in combination with erlotinib (arm B). A computed tomography (CT) scan at baseline and one at 4-12 weeks after inclusion was used to evaluate treatment response. Adverse events were registered during treatment and the subsequent 30 days. Health-related quality-of-life questionnaires were completed by the patients at baseline, weeks 2, 6, and 20. A total of 114 patients were included. Of the 74 patients with CT scans available for evaluation of treatment effect, there were no significant differences in tumor size reduction between the two groups: median 14.5% reduction in the control arm A and 17.0% in the erlotinib arm B (p = 0.68). Overall survival was not significantly different between the two treatment arms: 7.0 and 7.8 months in arm A and arm B, respectively (log-rank p = 0.32). There was no significant increase in adverse events in the experimental arm, other than what is expected from erlotinib treatment alone. Overall, patients reported similar quality of life in both treatment arms. Concurrent erlotinib and palliative thoracic RT for patients with advanced non-small cell lung cancer was well tolerated but did not improve the efficacy of the RT. ClinicalTrials.gov, identifier NCT02714530.

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/β-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia

The Wnt/β-catenin pathway’s significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/β-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/β-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active β-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish’s translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/β-catenin signaling.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

The differential prognostic implications of PD-L1 expression in the outcomes of Filipinos with EGFR-mutant NSCLC treated with tyrosine kinase inhibitors.

The tumor immune microenvironment influences tumor evolution in non-small cell lung cancer (NSCLC). Yet, the prognostic value of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor (EGFR)-mutant NSCLC remains controversial. Additionally, prognostic studies in Filipinos with EGFR-mutant NSCLC remain unexplored to this day. We prospectively studied the outcomes of EGFR-mutant NSCLC in Filipino cohort, and retrospectively verified the survival trend using The Cancer Genome Atlas (TCGA) cohort. Kaplan-Meier method and generalized linear regression were used to assess survival. Expression and DNA methylation of cluster of differentiation 274 (CD274, gene that codes for PD-L1) were examined from TCGA tumor profiles. Pearson's correlation was used to correlate PD-L1 expression with outcomes associated with occurrence of EGFR mutations, tyrosine kinase inhibitor (TKI) types, and programmed cell death protein 1 (PD-1) expression. Proteome network analysis was used to examine the correlation between drug resistance and PD-L1. PD-L1 positivity was associated with significantly longer progression-free survival (PFS; P=0.0096) but had a significantly contrasting influence in the overall survival (OS; P=0.0011). PD-L1 positivity (in both protein and RNA) was associated with longer median OS (mOS) in exon21 L858R, whereas, negativity was associated with longer mOS in exon19 deletion (exon19del). Stratification (high, low, negative) of PD-L1 expression lacked significant prognostic value (all P>0.05). PD-L1/CD274 expression (P<0.05) and DNA methylation (P<0.001) vary significantly among NSCLC subtypes and in different disease stages. Erlotinib treatment produced the longest median progression-free survival (mPFS; 874 days) relative to other EGFR-TKIs (137-311 days). PD-L1 lacked a significant correlation with EGFR-TKIs. Consistent with the immune-regulation activities of PD-1, higher expression leads to relatively shorter mOS. PD-1 correlated positively with PD-L1 expression and occurrence of exon21 L858R. PD-L1 differentially influenced the outcomes of Filipinos with EGFR-mutant NSCLC. NSCLC subtypes, disease stage, and PD-1 expression may impact the collective outcomes associated with PD-L1 and EGFR-sensitizing mutations.

A protocol of a single arm, prospective, open-label, multicenter, phase II study of ramucirumab and erlotinib in treatment-naïve non-small cell lung cancer patients with EGFR mutation and brain metastases (SPIRAL-BRAIN study).

The combination of erlotinib, a first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and ramucirumab, an anti-vascular endothelial growth factor receptor (VEGFR) antibody, is one of the most effective treatments for patients with non-small cell lung cancer (NSCLC) and EGFR mutation. However, little is known about the safety and efficacy of this combination treatment for patients with brain metastases. This single arm, prospective, open-label, multicenter, phase II study will recruit 32 NSCLC patients with EGFR mutation (except for T790M mutation) and brain metastases (asymptomatic or mild symptoms). Patients will be treated with erlotinib at a dose of 150 mg/body once daily and ramucirumab at a dose of 10 mg/kg once every 2 weeks. The primary endpoint is intracranial overall response rate (iORR) and the secondary endpoints are intracranial disease control rate, intracranial progression-free survival (iPFS), extracranial ORR, extracranial PFS, ORR, overall PFS, overall survival (OS), and safety. The planned number of enrollments was calculated based on a one-sample binomial test (normal approximation) with a two-sided α level of 5% and 80% power, assuming that the expected iORR is 65% and the iORR threshold is 40%. A prospective study to confirm the safety and efficacy of the combined erlotinib plus ramucirumab treatment for NSCLC patients with EGFR mutation and brain metastases is ongoing. Japan Registry of Clinical Trials, jRCTs051220059.

Abstract 4712: Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized

Abstract A close association between active fibroblastic foci (desmoplasia) and distant metastasis in the lung adenocarcinoma (LUAD) has been established decades ago and yet, the pro- and anti-tumor properties of cancer-associated fibroblasts (CAFs) are still being resolved in the hope of preventing LUAD progression. To better understand the role of stromal cells in progression, there is a growing interest in 3D patient-derived models for their translational and throughput advantages. In parallel, the emergence of multiplexed spatial biology platforms is advancing translational oncology forward by providing a more global understanding of the tumor microenvironment (TME). Despite the enthusiasm for these new technologies, there are no study showing the applicability and significance of using multiplexed spatial biology to characterized 3D patient-derived models. Here, we modeled spatial heterogeneity by assembling EGFR mutated patient-derived organoids (PDOs) with fibroblasts harvested from the tumor edge (tumor-adjacent fibroblasts; TAFs) or tumor core (tumor core fibroblasts; TCFs) referred here as tumor-stroma assembloids. Using these assembloids, we generated an extensive dataset with CODEX imaging technology and used a quantitative approach to dissect tumor-stroma crosstalk. Our results show that TAFs expressing the myofibroblast marker aSMA were strictly found at the periphery of the hydrogel dome containing the assembloid, while aSMA- fibroblasts were found strictly in the center. In contrast, we did not observe a similar spatial organization in TCF assembloids. Next, we used CELESTA, an unsupervised machine learning algorithm that uses prior knowledge of the cell expression and spatial information to dissect cell heterogeneity, then performed co-localization analyses. We also leveraged the EGFR mutation status of the PDOs and observed the protective effect of the fibroblasts from erlotinib. Both assembloid conditions showed drastically different tumor-stroma spatial changes including gains and losses of colocalizations even though erlotinib did not significantly change the subpopulation fractions, cell heterogeneity, nor cell density. The top tumor-stroma colocalization pairs that persisted after erlotinib involved either myofibroblasts and PanCK-Vim+MUC1+ cancer cells or were found in the center of the assembloids, shielded by a stromal barrier of myofibroblasts. Taken together, our results show that the assembloids undergo drastic spatial reorganization following erlotinib treatment even though subpopulation fractions and cell heterogeneity remain unchanged. This study provided new insights on tumor-stroma crosstalk in the LUAD and motivates the use 3D patient-derived models coupled with multiplex imaging to answer complex spatial biology questions. Citation Format: Gina Bouchard, Irene Li, Weiruo Zhang, Sylvia Plevritis. Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized. [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 4712.

A novel transcription factor-based signature to predict prognosis and therapeutic response of hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most common aggressive malignancies with increasing incidence worldwide. The oncogenic roles of transcription factors (TFs) were increasingly recognized in various cancers. This study aimed to develop a predicting signature based on TFs for the prognosis and treatment of HCC. Methods: Differentially expressed TFs were screened from data in the TCGA-LIHC and ICGC-LIRI-JP cohorts. Univariate and multivariate Cox regression analyses were applied to establish a TF-based prognostic signature. The receiver operating characteristic (ROC) curve was used to assess the predictive efficacy of the signature. Subsequently, correlations of the risk model with clinical features and treatment response in HCC were also analyzed. The TF target genes underwent Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, followed by protein-protein-interaction (PPI) analysis. Results: A total of 25 differentially expressed TFs were screened, 16 of which were related to the prognosis of HCC in the TCGA-LIHC cohort. A 2-TF risk signature, comprising high mobility group AT-hook protein 1 (HMGA1) and MAF BZIP transcription factor G (MAFG), was constructed and validated to negatively related to the overall survival (OS) of HCC. The ROC curve showed good predictive efficiencies of the risk score regarding 1-year, 2-year and 3-year OS (mostly AUC >0.60). Additionally, the risk score independently predicted OS for HCC patients both in the training cohort of TCGA-LIHC dataset (HR = 2.498, p = 0.007) and in the testing cohort of ICGC-LIRI-JP dataset (HR = 5.411, p < 0.001). The risk score was also positively correlated to progressive characteristics regarding tumor grade, TNM stage and tumor invasion. Patients with a high-risk score were more resistant to transarterial chemoembolization (TACE) treatment and agents of lapatinib and erlotinib, but sensitive to chemotherapeutics. Further enrichment and PPI analyses demonstrated that the 2-TF signature distinguished tumors into 2 clusters with proliferative and metabolic features, with the hub genes belonging to the former cluster. Conclusion: Our study identified a 2-TF prognostic signature that indicated tumor heterogeneity with different clinical features and treatment preference, which help optimal therapeutic strategy and improved survival for HCC patients.

Decursin inhibits EGFR-ERK1/2 signaling axis in advanced human prostate carcinoma cells.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.