An epigenetic switch regulates the ontogeny of AXL positive/EGFR-TKI resistant cells by modulating miR-335 expression

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. Many lung cancer patients will develop resistance to chemotherapeutics. In the context of non-small cell lung cancers harboring EGFR oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive Erlotinib resistance in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a sub-population of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines, and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells.

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An epigenetic switch regulates the ontogeny of AXL positive/EGFR-TKI resistant cells by modulating miR-335 expression

eLife ◽

10.7554/elife.66109 ◽

2021 ◽

Vol 10 ◽

Author(s):

Polona Safaric Tepes ◽

Debjani Pal ◽

Trine Lindsted ◽

Ingrid Ibarra ◽

Amaia Lujambio ◽

...

Keyword(s):

Cancer Cells ◽

Kinase Inhibitors ◽

Chemotherapeutic Agents ◽

Epigenetic Switch ◽

Lung Cancers ◽

Oncogenic Mutations ◽

Egfr Tyrosine Kinase Inhibitors ◽

A Cell ◽

Egfr Tki ◽

Resistant Cells

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. This is mainly due to the resistance that patients develop against chemotherapeutic agents over the course of treatment. In the context of non-small cell lung cancers (NSCLC) harboring EGFR oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive resistance to EGFR tyrosine kinase inhibitors such as Erlotinib and Osimertinib in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a sub-population of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines, and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR-oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells.

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Role of Omentin in Obesity Paradox in Lung Cancer

Cancers ◽

10.3390/cancers13020275 ◽

2021 ◽

Vol 13 (2) ◽

pp. 275

Author(s):

Sheetal Parida ◽

Sumit Siddharth ◽

Dipali Sharma

Keyword(s):

Lung Cancer ◽

Obesity Paradox ◽

Health Concern ◽

Case Control Studies ◽

Aberrant Expression ◽

Lung Cancers ◽

Lung Cancer Patients ◽

Significant Enrichment ◽

Normal Lungs ◽

Related Mortality

Lung cancer remains the second-most-common cancer worldwide and is associated with the highest number of cancer-related mortality. While tobacco smoking is the most important risk factor for lung cancer, many other lifestyles and occupational factors significantly contribute. Obesity is a growing global health concern and contributes to ~30% cancer-related mortality, but unlike other lifestyle diseases, lung cancer is negatively associated with obesity. We meta-analyzed multiple case-control studies confirming increased survival and better outcomes in overweight and obese lung cancer patients. Tumor heterogeneity analysis showed significant enrichment of adipocytes and preadipocytes in normal lungs compared to lung cancers. Interestingly, one of the understudied adipokine, omentin, was significantly and consistently lower in lung neoplasms compared to normal lungs. Omentin has been examined in relation to osteoarthritis, inflammatory bowel disease, cardiovascular diseases, diabetes, chronic liver disease, psoriasis and some other cancers. Aberrant expression of omentin has been reported in solid tumors; however, little is known about its role in lung cancer. We found omentin to be consistently downregulated in lung cancers, and it exhibited a negative correlation with important transcription factors FOXA1, EN1, FOXC1 and ELK4. We, therefore, suggest that omentin may serve as a prognostic factor in lung cancer and explain the “obesity paradox” in lung cancer.

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Comprehensive cancer genomics-based screening of new therapeutic targets and biomarkers for lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e21031 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e21031-e21031

Author(s):

Yataro Daigo ◽

Atsushi Takano ◽

Yusuke Nakamura

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Cancer Patients ◽

Therapeutic Target ◽

Cancer Genomics ◽

Cancer Biomarkers ◽

Lung Cancer Cells ◽

Lung Cancers ◽

Lung Cancer Patients ◽

Target Molecules

e21031 Background: Since the clinical outcome of advanced lung cancer patients is still poor after standard therapies, development of new anti-cancer drugs with minimum risk of adverse effects and cancer biomarkers for precision medicine is urgently required. Methods: We have been screening new therapeutic target molecules and molecular biomarkers for lung cancers as follows; i) To identify overexpressed genes in lung cancers by the gene expression profile analysis, ii) To verify the target genes for their scarce expression in normal tissues, iii) To validate the clinicopathologic importance of their protein expression by tissue microarray covering 263 lung cancers, and iv) To confirm their function for the growth and/or invasive ability of the lung cancer cells by siRNAs and gene transfection assays. Results: We identified dozens of candidate target molecules and selected a gene encoding protein with a GAP domain, LAPG1 (lung cancer-associated protein with Gap domain 1). Immunohistochemical analysis showed that LAPG1 expression was observed in 69.9% of lung cancers. Moreover positivity of LAPG1 expression was associated with poor prognosis of lung cancer patients. Knockdown of LAPG1 expression by siRNAs suppressed growth of lung cancer cells. Introduction of LAPG1 increased the invasive activity of mammalian cells, indicating that LAPG1 could be a prognostic biomarker and therapeutic target for lung cancers. Conclusions: Comprehensive cancer genomics-based screening could be useful for selection of new cancer biomarkers and molecular targets for developing small molecules, antibodies, nucleic acid drugs, and immunotherapies.

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Systematic approach for development of new immunotherapeutics for lung cancers through cancer genomics analysis.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3092 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3092-3092

Author(s):

Yataro Daigo ◽

Atsushi Takano ◽

Yusuke Nakamura

Keyword(s):

Lung Cancer ◽

Phase I ◽

Cancer Cells ◽

Cancer Patients ◽

Clinical Studies ◽

Critical Role ◽

Phase I Clinical Trials ◽

Lung Cancers ◽

Lung Cancer Patients ◽

High Immunogenicity

3092 Background: Oncoantigens are defined to be proteins that are very specifically expressed in cancer cells and that have the oncogenic activity and high immunogenicity, and are considered to be promising targets for immunotherapy such as therapeutic cancer vaccines. Methods: We have established a strategy as follows to identify new oncoantigens; i) screening of highly transactivated genes in the majority of 120 lung cancers using cDNA microarray representing 27,648 genes coupled with enrichment of tumor cells by laser microdissection, ii) verification of no expression of each candidate gene in normal tissues by northern-blot analysis, iii) validation of the clinicopathological significance of its high level of expression with tissue microarray containing 300 lung cancers, iv) verification of a critical role of each gene in the growth or invasiveness of cancer cells by RNAi and cell growth/invasion assays, v) screening of the epitope peptides recognized by HLA-A*0201- or A*2402-restricted cytotoxic T lymphocyte (CTL). We conducted phase I clinical trials of these therapeutic peptide vaccines for lung cancer patients. Results: We identified 35 oncoantigens and screened dozens of 10-amino-acid peptides, each of which corresponded to a part of TTK, LY6K, IMP-3, CDCA1, KIF20A, CDC45L, and FOXM1, and was a candidate to be presented on the surface of HLA-A*0201 or HLA-A*2402 that induced in vitro CTL response. Phase I clinical studies indicated that five epitope peptides could strongly induce the CTL activity in cancer patients. For example, we conducted a phase I study for HLA-A*2402-positive, advanced non-small cell lung cancer patients who failed to standard therapy, using the combination of 1, 2 or 3 mg/body of each peptides from LY6K, CDCA1, and KIF20A mixed with adjuvant once a week. This cancer vaccine therapy demonstrated tolerability and had very high immunogenicity of even 1 mg/body dose to induce antigen-specific CTLs in cancer patients. Conclusions: Through systematic genomics-based approach and clinical study, we have identified five epitope peptides, which could induce CTLs very effectively in cancer patients, and therefore it warrants further clinical studies. Clinical trial information: NCT01069575.

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The predictive value of uncommon EGFR mutation in patients with non-small-cell lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.9028 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 9028-9028

Author(s):

Haiyan Tu ◽

Ee Ke ◽

Yue-Li Sun ◽

Ming-Ying Zheng ◽

Jin-Ji Yang ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Patients ◽

Growth Factor Receptor ◽

Small Cell ◽

Egfr Mutations ◽

Small Cell Lung ◽

Lung Cancers ◽

Lung Cancer Patients ◽

Exon 20 ◽

Egfr Tki

9028 Background: Non-small-cell lung cancers with uncommon epidermal growth factor receptor ( EGFR) mutations are regarded as a heterogeneous group with variable responses to EGFR-targeted drugs. Here we designed this retrospective study to describe the epidemiology and clinical outcomes of uncommon EGFR mutations in a Chinese cohort of lung cancer patients. Methods: Between June 2007 and June 2014, 5363 lung cancer patients whose EGFR genotyping was performed successfully at Guangdong Lung Cancer Institute (GLCI, Guangzhou, China) were screened. 1837 patients were included in the epidemiological analysis. The clinical outcome was analyzed in 97 advanced-stage patients harboring uncommon EGFR mutations with follow-up data. Results: 218 patients harbored uncommon EGFR mutations, making up 11.9% of all cancers with documented EGFR mutations. Compared with common mutants, those with uncommon mutations were more commonly found in smokers and male patients. The most frequently detected uncommon mutations were exon 20 insertions, G719X mutations and L858R complex mutations, occurring in 30.7%, 21.1% and 17.0% of all EGFR-uncommon-mutation cases. G719X and L858R complex mutations were associated with similar benefit from EGFR-TKI; median PFS was 15.2 (95% CI 8.7-21.7) and 11.6 (95% CI 3.6-19.6) months, respectively. T790M or 20INS was associated with a poorer EGFR-TKI response; median PFS was 1.0 (95% CI 0.0-2.2) and 3.0 (95% CI 1.3-4.7) months, respectively. Of note, two patients with 23% and 65% tumor shrinkages had N771_P772insN and H773_V774insQ, with PFS of 5.7 and 6.1 months respectively. Conclusions: Favorable responses were observed in specific subtypes including complex L858R and G719X, and our results suggested first-line EGFR-TKI should be preferable in such patients.

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Mechanism of Xu Li’s Experiential Prescription for the Treatment of EGFR-Positive NSCLC

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/8787153 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Bin Xu ◽

Haitao Zhang ◽

Yuchao Wang ◽

Shuran Huang ◽

Li Xu

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Small Cell ◽

Drug Resistant ◽

M2 Macrophages ◽

Cellular Activity ◽

Small Cell Lung ◽

Associated Proteins ◽

Egfr Tki ◽

Resistant Cells

The non-small-cell lung cancer (NSCLC) is the most common lung cancer which seriously threatens the human health. Xu Li’s experiential prescription (XLEP) can treat the NSCLC. However, whether XLEP can regulate the autophagy in the EGFR-positive NSCLC still remains unknown. We found that the cellular activity of drug-resistant cells and sensitive cells were all decreased in the TCM group and TCM + Gef group. The expression of autophagy-associated proteins (mTOR and Beclin1-Vps34) in drug-resistant cells was decreased in the TCM group, while the expression of autophagy-associated proteins in sensitive cells was all decreased in the TCM + Gef group. The ratio of M1/M2 macrophages was increased when IL-4-induced RAW264.7 was treated with TCM. TCM treatment promoted the expression of CCL2 and CCL3 while it downregulated the CCL22 level among A549, H1975, and PC9 cells. The expression of TNF-α and IL-6 was increased, and the expression of IL-10 and TGF-β was decreased in IL-4-induced RAW264.7 cells treated with TCM. And, TCM treatment also decreased the expression of Fizz1 and TGM2. In conclusion, this study indicated that XLEP could suppress the proliferation of EGFR-TKI-resistant cancer cells and increase the ratio of M1/M2 macrophages by inhibiting autophagy to treat the drug-resistant EGFR-positive NSCLC.

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Inhibition of miR-185-3p Confers Erlotinib Resistance Through Upregulation of PFKL/MET in Lung Cancers

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.677860 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ke Li ◽

Xinling Zhu ◽

Conghu Yuan

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Lung Cancer Cells ◽

Cell Counting ◽

Luciferase Assay ◽

Lung Cancers ◽

Tki Resistance ◽

Egfr Tki

Erlotinib (ER), as an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has a significant therapeutic effect in lung cancers. However, EGFR TKI resistance inevitably occurs after treatment for approximately 12 months, which weakens its antitumor effect. Here, we identified miR-185-3p as a significantly downregulated microRNA responsible for acquired EGFR TKI resistance in cells and patients with lung cancer. qRT-PCR and Western Blot were performed to determine the relative expression of miR-185-3p in ER-resistant tumor tissues and cells. The viability and apoptosis of lung cancer cells were evaluated by Cell Counting Kit-8 (CCK8) assay and flow cytometry, respectively. The binding between miR-185-3p and liver-type phosphofructokinase (PFKL) was verified by dual luciferase assay. It was found that overexpression of miR-185-3p conferred ER sensitivity in lung cancer cell lines. MiR-185-3p was downregulated in ER-resistant lung cancer cells (H1299/ER and A549/ER). MiR-185-3p inhibited proliferation and induced cell apoptosis in ER-resistant cells. Mechanistically, miR-185-3p downregulation contributed to ER resistance through upregulating the PFKL. Moreover, Mesenchymal to epithelial transition (MET) oncoprotein promoted EGFR-TKI resistance by regulating miR-185-3p and PFKL. These findings revealed a novel mechanism in which downregulation of miR-185-3p may induce overexpression of PFKL and MET and confer ER resistance in lung cells. Combination of PFKL/MET inhibitors and EGFR TKIs could be a rational therapeutic approach for lung cancer patients with EGFR mutation.

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The Root Extract of Scutellaria baicalensis Induces Apoptosis in EGFR TKI-Resistant Human Lung Cancer Cells by Inactivation of STAT3

International Journal of Molecular Sciences ◽

10.3390/ijms22105181 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5181

Author(s):

Hyun-Ji Park ◽

Shin-Hyung Park ◽

Yung-Hyun Choi ◽

Gyoo-Yong Chi

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Human Lung ◽

Scutellaria Baicalensis ◽

Lung Cancer Cells ◽

Human Lung Cancer ◽

Root Extract ◽

Egfr Tki ◽

Resistant Cells

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in managing lung cancer. The root of Scutellaria baicalensis (SB) traditionally used for fever clearance and detoxification possesses various bioactivities including anticancer effects. The purpose of this study was to investigate whether SB exhibited anticancer activity in EGFR TKI-resistant lung cancer cells and to explore the underlying mechanism. We used four types of human lung cancer cell lines, including H1299 (EGFR wildtype; EGFR TKI-resistant), H1975 (acquired TKI-resistant), PC9/ER (acquired erlotinib-resistant), and PC9/GR (acquired gefitinib-resistant) cells. The ethanol extract of SB (ESB) decreased cell viability and suppressed colony formation in the four cell lines. ESB stimulated nuclear fragmentation and the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3. Consistently, the proportion of sub-G1 phase cells and annexin V+ cells were significantly elevated by ESB, indicating that ESB induced apoptotic cell death in EGFR TKI-resistant cells. ESB dephosphorylated signal transducer and activator of transcription 3 (STAT3) and downregulated the target gene expression. The overexpression of constitutively active STAT3 reversed ESB-induced apoptosis, suggesting that ESB triggered apoptosis in EGFR TKI-resistant cells by inactivating STAT3. Taken together, we propose the potential use of SB as a novel therapeutic for lung cancer patients with EGFR TKI resistance.

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Integrated genomics-based approach to identify new therapeutic targets and cancer biomarkers for lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e13019 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e13019-e13019

Author(s):

Yataro Daigo ◽

Atsushi Takano ◽

Yusuke Nakamura

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Cancer Patients ◽

Mammalian Cells ◽

Therapeutic Targets ◽

Cancer Biomarkers ◽

Lung Cancer Cells ◽

Lung Cancers ◽

Lung Cancer Patients ◽

Integrated Genomics

e13019 Background: Because the number of lung cancer patients who show good response to standard therapies is still limited, development of new anti-cancer agents with minimum risk of adverse effects and highly sensitive molecular biomarkers is urgently required. Methods: We have been screening novel therapeutic targets and their companion biomarkers for lung cancer as follows; i) To identify up-regulated genes in lung cancers by the gene microarray analysis, ii) To verify the candidate genes for their low expression in normal organs, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering hundreds of lung cancers, and iv) To verify their function for the growth of lung cancer cells by siRNAs. Results: We identified dozens of candidate oncoproteins and selected a serine/threonine kinase LASK2 (lung cancer-associated kinase 2). Immunohistochemical analysis showed that strong LASK2 positivity was an independent prognostic factor for non-small cell lung cancer patients (P < 0.0001). Suppression of LASK2 expression by its siRNAs inhibited proliferation of lung cancer cells. Introduction of LASK2 in mammalian cells also enhanced cellular growth in vitro and in mice model. Induction of LASK2 appeared to increase the levels of phosphorylation of oncogenic signal proteins for lung cancer. The data indicate that LASK2 is a prognostic biomarker and therapeutic target for lung cancers. Conclusions: Integrated genomics-based approach could facilitate the development of new cancer biomarkers as well as therapeutic targets for small molecules, monoclonal antibodies, nucleic acid drugs, and immunotherapies.

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Autophagy determines osimertinib resistance through regulation of stem cell-like properties in EGFR-mutant lung cancer

10.1101/330092 ◽

2018 ◽

Author(s):

Li Li ◽

Yubo Wang ◽

Lin Jiao ◽

Caiyu Lin ◽

Conghua Lu ◽

...

Keyword(s):

Lung Cancer ◽

Drug Resistance ◽

Stem Cell ◽

Lung Cancer Patients ◽

Knock Down ◽

Egfr Mutant ◽

Egfr Tki ◽

Autophagy Inhibition ◽

Resistant Cells

ABSTRACTDrug resistance to Osimertinib, a 3rd-generation EGFR-TKI is inevitable. Autophagy plays a contradictory role in resistance of 1stand 2ndgeneration EGFR-TKI, and its significance in osimertinib resistance is much less clear. We therefore investigated whether autophagy determines osimertinib resistance. First, osimertinib induced autophagy to a much greater extent than that of gefitinib, and autophagy inhibition further increased osimertinib efficacy. Next, enhanced autophagy was found in osimertinib resistant cells and autophagy inhibition partially reversed osimertinib resistance. Enhanced stem-cell like properties were found in resistant cells, and siRNA-knock down ofSOX2orALDH1A1reversed osimertinib resistance. Of note, autophagy inhibition or siRNA-knock down of Beclin-1 decreased expression of SOX2 and ALDH1A1 and stem-cell like properties. Next, autophagy inhibition and osimertinib in combination effectively blocked tumor growth in xenografts, which was associated with decreased autophagy and stem cell-like propertiesin vivo. Finally, enhanced autophagy was found in lung cancer patients with resistance to osimertinib. In conclusion, the current study delineates a previously unknown function of autophagy in determining osimertinib resistance through promoting stem-cell like properties.

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