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• W4376598246 abstract "Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells in vitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations in vivo. Mechanically, 35d triggers heat shock protein 70–mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease. Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells in vitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations in vivo. Mechanically, 35d triggers heat shock protein 70–mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease. Lung cancer is the leading cause of cancer-related deaths worldwide (1Bade B.C. Dela Cruz C.S. Lung cancer 2020: epidemiology, etiology, and prevention.Clin. Chest Med. 2020; 41: 1-24Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar), with non–small cell lung cancers (NSCLCs) accounting for around 85 to 90% of cases. Of these NSCLCs, lung adenocarcinomas (LUADs) and lung squamous cell carcinoma (LUSC) make up ∼55% and ∼35%, respectively. Genetic mutation on epidermal growth factor receptor (EGFR) causes aberrant kinase activation in ∼50% of LUADs in Asian patients and ∼15% in Western population (2Shaurova T. Zhang L. Goodrich D.W. Hershberger P.A. Understanding lineage plasticity as a path to targeted therapy failure in EGFR-mutant non-small cell lung cancer.Front. Genet. 2020; 11: 281Crossref PubMed Scopus (37) Google Scholar, 3Bartholomew C. Eastlake L. Dunn P. Yiannakis D. EGFR targeted therapy in lung cancer; an evolving story.Respir. Med. Case Rep. 2017; 20: 137-140Crossref PubMed Scopus (17) Google Scholar). EGFR is known to play a critical role in maintaining tumor cell survival through its downstream signaling that rely on its kinase activity (4Cai W.Q. Zeng L.S. Wang L.F. Wang Y.Y. Cheng J.T. Zhang Y. et al.The latest battles between EGFR monoclonal antibodies and resistant tumor cells.Front. Oncol. 2020; 10: 1249Crossref PubMed Scopus (59) Google Scholar, 5Hsu J.L. Hung M.C. The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer.Cancer Metastasis Rev. 2016; 35: 575-588Crossref PubMed Scopus (185) Google Scholar, 6Chen C.H. Wang B.W. Hsiao Y.C. Wu C.Y. Cheng F.J. Hsia T.C. et al.PKCdelta-mediated SGLT1 upregulation confers the acquired resistance of NSCLC to EGFR TKIs.Oncogene. 2021; 40: 4796-4808Crossref PubMed Scopus (6) Google Scholar). Tyrosine kinase inhibitors (TKIs) have been effective in treating EGFR-mutant patients, but resistance develops after 10 to 18 months, and recurrent diseases currently lack effective therapies. Several TKI-resistant mechanisms have been identified in EGFR-mutant NSCLCs, including upregulation of other receptor tyrosine kinases (RTKs) (7Del Re M. Crucitta S. Gianfilippo G. Passaro A. Petrini I. Restante G. et al.Understanding the mechanisms of resistance in EGFR-positive NSCLC: from tissue to liquid biopsy to guide treatment strategy.Int. J. Mol. Sci. 2019; 20: 3951Crossref PubMed Scopus (49) Google Scholar, 8Chen M.K. Hung M.C. Regulation of therapeutic resistance in cancers by receptor tyrosine kinases.Am. J. Cancer Res. 2016; 6: 827-842PubMed Google Scholar) and development of additional EGFR mutations, such as C797S (9Rotow J. Bivona T.G. Understanding and targeting resistance mechanisms in NSCLC.Nat. Rev. Cancer. 2017; 17: 637-658Crossref PubMed Scopus (550) Google Scholar, 10Minari R. Bordi P. Tiseo M. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance.Transl. Lung Cancer Res. 2016; 5: 695-708Crossref PubMed Scopus (142) Google Scholar, 11Piotrowska Z. Niederst M.J. Karlovich C.A. Wakelee H.A. Neal J.W. Mino-Kenudson M. et al.Heterogeneity underlies the emergence of EGFRT790 wild-type clones following treatment of T790M-positive cancers with a third-generation EGFR inhibitor.Cancer Discov. 2015; 5: 713-722Crossref PubMed Scopus (383) Google Scholar). Interestingly, the mechanisms of RTK-mediated TKI resistance have been linked to EGFR protein expression per se, independent of EGFR kinase activity (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Upon TKI treatment, the upregulated RTKs in resistant tumors form heterodimers with TKI-inactive EGFR and activate downstream survival signaling (hereafter referred to as TKI-insensitive EGFR signaling). The TKI-insensitive EGFR signaling dominate the tumors upon TKI and contribute to heterogeneous TKI resistance and tumorigenesis (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Weihua Z. Tsan R. Huang W.C. Wu Q. Chiu C.H. Fidler I.J. et al.Survival of cancer cells is maintained by EGFR independent of its kinase activity.Cancer Cell. 2008; 13: 385-393Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Additionally, the acquisition of EGFR C797S mutations after TKI treatment also results in currently untreatable disease (7Del Re M. Crucitta S. Gianfilippo G. Passaro A. Petrini I. Restante G. et al.Understanding the mechanisms of resistance in EGFR-positive NSCLC: from tissue to liquid biopsy to guide treatment strategy.Int. J. Mol. Sci. 2019; 20: 3951Crossref PubMed Scopus (49) Google Scholar). Despite this, C797S-positive tumors may still rely on EGFR kinase activity for survival, as evidenced by their sensitivity to next-generation EGFR kinase inhibitors in preclinical models (10Minari R. Bordi P. Tiseo M. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance.Transl. Lung Cancer Res. 2016; 5: 695-708Crossref PubMed Scopus (142) Google Scholar, 14To C. Jang J. Chen T. Park E. Mushajiang M. De Clercq D.J.H. et al.Single and dual targeting of mutant EGFR with an allosteric inhibitor.Cancer Discov. 2019; 9: 926-943Crossref PubMed Scopus (158) Google Scholar). However, patients with C797S tumors still lack effective treatment in clinical. Therefore, pharmacological inhibitions of the TKI-insensitive EGFR signaling and the EGFR C797S by reducing EGFR protein expression may offer therapeutic opportunities for NSCLC patients with acquired TKI resistance. Here, we developed a small molecule 35d which activates heat shock protein (hsp) 70–mediated lysosome pathway, degrading EGFR protein, and inhibiting TKI-insensitive EGFR signaling. 35d alone or with TKIs suppressed tumor growth in TKI-resistant LUAD tumors, including those with variant resistant mechanisms such as a common factor, nuclear PKCδ (found in >40% of the resistant LUADs) (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and EGFR C797S mutation (found in ∼15% of the resistant LUADs) (7Del Re M. Crucitta S. Gianfilippo G. Passaro A. Petrini I. Restante G. et al.Understanding the mechanisms of resistance in EGFR-positive NSCLC: from tissue to liquid biopsy to guide treatment strategy.Int. J. Mol. Sci. 2019; 20: 3951Crossref PubMed Scopus (49) Google Scholar). These findings suggest that 35d could be a potential therapy for EGFR-mutant LUADs resistant to TKI. EGFR, an important RTK and oncogene driver, plays a vital role in NSCLC (15Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. et al.Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (10115) Google Scholar). However, inhibition of EGFR kinase activity by clinical TKIs only benefits to a subpopulation of NSCLC patients (16Shepherd F.A. Tsao M.S. Unraveling the mystery of prognostic and predictive factors in epidermal growth factor receptor therapy.J. Clin. Oncol. 2006; 24 (author reply 1220-1211): 1219-1220Crossref PubMed Scopus (90) Google Scholar, 17Tomasello C. Baldessari C. Napolitano M. Orsi G. Grizzi G. Bertolini F. et al.Resistance to EGFR inhibitors in non-small cell lung cancer: clinical management and future perspectives.Crit. Rev. Oncol. Hematol. 2018; 123: 149-161Crossref PubMed Google Scholar). To systematically investigate whether TKI-insensitive EGFR signaling is critical for NSCLCs, we analyzed the EGFR expression by evaluating DNA copy number (CN) and mRNA expression levels in LUAD and LUSC cohorts from The Cancer Genome Atlas (TCGA) NSCLC dataset. We also determined whether EGFR CN and mRNA expression levels can predict patient survival. The patients with EGFR CN gain exhibited shorter overall survival (OS) than the patients with normal CN in LUAD subset (Fig. 1A, p = 0.0002). Consistently, higher EGFR mRNA expression in the tumors was associated with inferior OS of LUAD patients (Fig. 1B, p = 0.0404). Furthermore, a significantly positive correlation between EGFR gene CN and mRNA expression levels were found in LUAD tumors (Fig. 1C, p = 0.00291). However, in LUSC cohort, there were no correlation of EGFR CN alteration (Fig. 1D, p = 0.249) and mRNA levels (Fig. 1E, p = 0.448) with patient OS. The results were subsequently confirmed using Kaplan–Meier plotter mRNA RNA-seq dataset (18Nagy A. Munkacsy G. Gyorffy B. Pancancer survival analysis of cancer hallmark genes.Sci. Rep. 2021; 11: 6047Crossref PubMed Scopus (461) Google Scholar), where high EGFR mRNA expression associated with poor OS in LUAD (Fig. 1F, p = 0.011), but not in LUSC (Fig. 1G, p = 0.16). These data suggested that EGFR expression levels in the tumors are critical for patient survival in LUAD, but not in LUSC. While EGFR mutation status is commonly used as a therapeutic marker for TKIs in treating lung cancer, our findings indicated that the expression levels of EGFR in tumors are still critical for survival, especially in LUAD patients, suggesting a potential role of TKI-insensitive EGFR signaling in LUADs. Therefore, we investigated whether EGFR status determines the dependence of LUADs on TKI-insensitive EGFR signaling. Several LUAD cell lines with variant EGFR status and TKI response were treated with EGFR shRNA to block TKI-insensitive EGFR signaling through depleting EGFR expression (Fig. 1, H–M). Cell viability analysis showed that EGFR-WT H460 and H1299 cells were insensitive to both EGFR kinase inhibitor gefitinib (gef) and EGFR shRNA (Fig. 1, H and I), suggesting that TKI-insensitive EGFR signaling may be not essential for survival of these EGFR-WT LUAD cell lines. On the contrary, EGFR L858R-mutant H3255 cells highly sensitized to EGFR shRNA (Fig. 1, H and I), which are expected because the H3255 cells harboring EGFR-activating mutation sensitize to EGFR inhibitor. The EGFR additional C797S mutation was currently discovered as one of the major acquired resistance mechanisms to the third generation TKI, osimertinib (osi) (19Yang Z. Yang N. Ou Q. Xiang Y. Jiang T. Wu X. et al.Investigating novel resistance mechanisms to third-generation EGFR tyrosine kinase inhibitor osimertinib in non-small cell lung cancer patients.Clin. Cancer Res. 2018; 24: 3097-3107Crossref PubMed Scopus (299) Google Scholar), and such patients do not have any effective treatment in clinical. We next tested the effects of EGFR depletion in a patient-derived VGHT 11 cells harboring del19/T790M/C797S mutations on EGFR (20Lin C.Y. Huang K.Y. Lin Y.C. Yang S.C. Chung W.C. Chang Y.L. et al.Vorinostat combined with brigatinib overcomes acquired resistance in EGFR-C797S-mutated lung cancer.Cancer Lett. 2021; 508: 76-91Crossref PubMed Scopus (13) Google Scholar). We confirmed that the T790M/C797S-positive VGHT 11 cells were resistant to both first generation TKI gef and third generation TKI osi (Fig. 1, K and L). Importantly, we found that the cell growth of VGHT 11 cells was markedly suppressed by the treatment of EGFR shRNA (Fig. 1, K–M), suggesting a therapeutic potential for the untreatable C797S-mutant tumors. Previous study reported that EGFR-mutant H1650 cells that harbor intrinsically resistant mechanisms to TKIs through activation of PKCδ, AKT, and NF-kB pathways were susceptible to EGFR depletion (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 21Bivona T.G. Hieronymus H. Parker J. Chang K. Taron M. Rosell R. et al.FAS and NF-kappaB signalling modulate dependence of lung cancers on mutant EGFR.Nature. 2011; 471: 523-526Crossref PubMed Scopus (344) Google Scholar, 22Sos M.L. Koker M. Weir B.A. Heynck S. Rabinovsky R. Zander T. et al.PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR.Cancer Res. 2009; 69: 3256-3261Crossref PubMed Scopus (445) Google Scholar). All these results indicated that EGFR-mutant cells, but not EGFR WT cells we tested, may sensitize to EGFR depletion and suggested that targeting of TKI-insensitive EGFR signaling by reducing EGFR protein expression may be a therapeutic strategy for treating TKI-insensitive or TKI-acquired resistant patients with EGFR-mutant LUADs, which patients currently lack effective treatment in clinical. To convert the in vitro concept to practical practices in preclinical and in clinical, we looked for emerged candidate agents that exerted to reduce EGFR expression and have potential to further develop as anticancer drugs. We tended to focus on natural compounds as many of these compounds were generally tolerable in human. Text-mining screenings were performed to identify compound candidates that were showed to reduce EGFR expression in which we searched more than 2500 preclinical and clinical studies between 1995 and 2021 (Fig. 2A). Nine compound candidates were identified, which have been showed to reduce EGFR expression in cancer cells and were reproduced in at least two literatures (Fig. 2A, Tables 1 and S1). Further investigation of these compounds indicated that five of them have been tested in clinical trials (Fig. 2A and Table S2). Among these candidates, curcumin has been shown to reduce EGFR expression in several studies (Fig. 2A and Table 1) and has been used to treat malignant diseases, including lung cancers (23Lee J.Y. Lee Y.M. Chang G.C. Yu S.L. Hsieh W.Y. Chen J.J. et al.Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy.PLoS One. 2011; 6e23756Google Scholar, 24Chen P. Huang H.P. Wang Y. Jin J. Long W.G. Chen K. et al.Curcumin overcome primary gefitinib resistance in non-small-cell lung cancer cells through inducing autophagy-related cell death.J. Exp. Clin. Cancer Res. 2019; 38: 254Crossref PubMed Scopus (84) Google Scholar). Moreover, curcumin has been tested in more than 270 clinical trials including more than 25 phase 3 trials with well-tolerant doses (Table S2), indicating that curcumin is a highly safe agent in human. However, poor chemical stability, low water solubility, and low oral bioavailability remain major challenges for curcumin in the utilization as a therapeutic drug (25Liu W. Zhai Y. Heng X. Che F.Y. Chen W. Sun D. et al.Oral bioavailability of curcumin: problems and advancements.J. Drug Target. 2016; 24: 694-702Crossref PubMed Scopus (354) Google Scholar). We have previously modified and synthesized several series of diarylheptanoid derivatives based on chemical structure of curcumin (26Hsieh M.T. Chang L.C. Hung H.Y. Lin H.Y. Shih M.H. Tsai C.H. et al.New bis(hydroxymethyl) alkanoate curcuminoid derivatives exhibit activity against triple-negative breast cancer in vitro and in vivo.Eur. J. Med. Chem. 2017; 131: 141-151Crossref PubMed Scopus (20) Google Scholar). Among the derivatives, MTH-3 was the first to be synthesized and showed greater cell-growth inhibitory activities than curcumin against breast cancer cells (27Chang L.C. Hsieh M.T. Yang J.S. Lu C.C. Tsai F.J. Tsao J.W. et al.Effect of bis(hydroxymethyl) alkanoate curcuminoid derivative MTH-3 on cell cycle arrest, apoptotic and autophagic pathway in triple-negative breast adenocarcinoma MDA-MB-231 cells: an in vitro study.Int. J. Oncol. 2018; 52: 67-76PubMed Google Scholar). To evaluate whether these diarylheptanoid derivatives suppress EGFR expression and inhibit the tumor growth in distinct EGFR-mutant, TKI-resistant lung cancer models, we performed a growth inhibitory screening by treating curcumin, MTH-3, and 14 diarylheptanoid derivatives in EGFR-mutant H1650 cells (Fig. 2, B and C), which cells are known to be intrinsically resistant to EGFR kinase inhibition but sensitize to EGFR protein depletion (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We found that among the 14 diarylheptanoid derivatives, 22b, 36, 35a, 37, and 35d showed lower IC50 values than MTH-3 and curcumin, indicating their greater effects in killing H1650 cells (Fig. 2, B and C). Among these five derivatives, 22b showed relatively low solubility (data not shown), which may cause failure for drug development in the future, and was excluded for following analysis.Table 1Emerged natural compounds that reduce EGFR expression in cancer cellsCompoundTypes of cancer cellsDetection methodsCurcumin and its derivativesBladder: 253JB-V, KU7Breast: MDA-MB-231Colon: RKO, SW480Lung: CL1-5, A549, H1975; H226Nasopharyngeal: CNE-2Osteosarcoma: MG-63Pancreas: P34, PC14Skin: A431WBCucurbitacin BBreast: MDA-MB-231, SKBR3Cholangiocarcinoma: KKU-100Pancreatic: BxPC3, HPACWBDeguelinBreast cancer: MDA-MB-231HNSCC: SCC-4WB, IF(−)-Epigallocatechin-3-gallateBreast: MDA-MB-231, MCF-7Bronchial: BEAS-2BColorectal: Caco-2Esophageal: KYSE150, A431Head and neck: SACC-83Lung: A549WBHonokiol/liposomal honokiolBrain: U251, U87MGBronchial: 1117Head and neck: FaDu, SCC-1, Cal-33, KB-3-1Liver: Hep3B, HepG2Lung: PC-9, H1975, HCC827, H460, SPC-A1WB, IFPlumbagin and its derivativeLung: H460Pancreas: PANC1, BxPC3, ASPC1WBQuercetin/gold nanoparticles-conjugated quercetinBreast: MDA-MB-231, MCF-7Cervical: HeLaProstate: PC-3, LNCaPWBResveratrolEsophageal cancer: KYSE150, Eca109Glioma: C6WBSulforaphaneBreast cancer: MDA-MB-231, MDA-MB-468, T47DLung cancer: A549, H1975, H3255, PC9WBThe compounds in this table were showed to reduce EGFR expression in more than one publication.Abbreviation: IF, immunofluorescence analysis. Open table in a new tab The compounds in this table were showed to reduce EGFR expression in more than one publication. Abbreviation: IF, immunofluorescence analysis. We have modeled acquired resistance to gef from TKI-sensitive, EGFR-mutant LUAD HCC827 cells and followed by a single cell isolation (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Several gef-resistant (GR) clones have been generated, and all the GR clones are highly resistant to gef and afatinib compared to their parental cells. Each GR clone expresses several and distinguished resistant mechanisms independently of EGFR T790M mutations (12Lee P.C. Fang Y.F. Yamaguchi H. Wang W.J. Chen T.C. Hong X. et al.Targeting PKCdelta as a therapeutic strategy against heterogeneous mechanisms of EGFR inhibitor resistance in EGFR-mutant lung cancer.Cancer Cell. 2018; 34: 954-969.e4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this study, we evaluated anticancer effects of the four derivatives in our acquired resistance models. The results indicated that all the GR cells were more sensitive to these four diarylheptanoid derivatives than MTH-3 (Fig. 2D). To further evaluate the effects of these compounds, cytotoxicity analysis of the four diarylheptanoid derivatives and MTH-3 were performed in H1650 and in a well-characterized gef-resistant H1975 cell model with EGFR T790M mutation (Fig. 2, E and F, respectively). Among the four derivatives, 35d (Fig. 2G) is the most effective to inhibit cell growth in H1650 and H1975 cells. Our data from intrinsically TKI-resistant cell line, acquired resistant clones, and T790M-positive cell line suggest therapeutic potential of 35d for these clinically irremediable LUADs. Moreover, we noticed that the IC50 of curcumin, MTH-3, and 35d are dropping from >16, 3.6 ∼ 6.6, to 0.5 ∼ 2.5 μM, respectively, in all the TKI-resistant LUAD cells we tested (Fig. 2C), indicating that our chemical modification strategies effectively improve their anticancer activities against EGFR-mutant, TKI-resistant LUAD cells. To explore the specific mechanisms in TKI-resistant cells treated with 35d, globally transcriptional analysis by RNA-seq was performed in 35d- and curcumin-treated GR6 cells (Fig. 3, A and B). Principal component analyses of the RNA-seq data revealed global changes by 35d treatment in the transcriptome (Fig. 3C). Interestingly, five of top ten gene sets were related to aspects of heat responses, such as response to heat, response to unfolded protein, and cellular response to heat (Fig. 3C, red). Three of them were apoptotic signaling pathways, suggesting that 35d may induce cancer cell death through apoptosis (Fig. 3C). To identify the molecular events that trigger the heat responses in the 35d-treated cells, we revisited our RNA-seq data. We found that several HSP70 family genes were involved in the heat response gene sets (Table S3). Further analysis of all HSP70 family genes in the RNA-seq data revealed that mRNA expression levels of HSPA1A, HSPA1B, HSPA6, and HSPA8 were >2-fold increased in response to 35d (Fig. 3D, left). Interestingly, curcumin at the same concentration did not affect any of these HSP70 gene expressions (Fig. 3D right). To investigate the clinical significance of the HSP70 family genes in LUADs, we analyzed a human LUAD dataset, containing crucial patient information such as tumor EGFR mutation status, gene expression profile, patient TKI-treatment history, and OS (28Chen J. Yang H. Teo A.S.M. Amer L.B. Sherbaf F.G. Tan C.Q. et al.Genomic landscape of lung adenocarcinoma in East Asians.Nat. Genet. 2020; 52: 177-186Crossref PubMed Scopus (188) Google Scholar). Using this dataset, we categorized the LUAD patients into three cohorts based on EGFR status and TKI treatment (EGFR-mutant patients with TKI treatment, EGFR-mutant patients without TKI treatment, and EGFR WT patients without TKI treatment). We then correlated the mRNA expression levels of all the HSP70 family genes with patient OS in each cohort (Table S4). Importantly, among the 15 HSP70 family genes analyzed, our results revealed that higher HSPA1B significantly correlated with longer OS in the EGFR-mutant patients with TKI treatment (p = 0.011; Table S4 and Fig. 3E). However, this correlation was not observed in the EGFR-mutant patients without TKI and the EGFR WT patients (p = 0.998 and 0.964, respectively; Table S4). These results indicated that 35d upregulated HSP70 genes in the EGFR-mutant, TKI-resistant cells and suggested high HSPA1B expression in the tumors may be clinically beneficial for only the EGFR-mutant LUAD patients who underwent TKI treatment. Our data indicate that the EGFR-mutant LUAD with TKI resistance is a unique subtype among NSCLCs that relies on the EGFR protein expression per se for survival (Fig. 1). Our data has shown that a synthesized, small molecule curcumin derivative 35d is highly effective in inhibiting the growth of these cells (Fig. 2). To better understand how 35d achieves these effects, we investigated whether it works by reducing EGFR expression. The result indicated that 35d dose dependently reduced EGFR protein expression in various EGFR-mutant, TKI-resistant cells including GR6, GR8, GR10 (Fig. 4, A and B left), H1650, H1975 (Fig. 4C), and in EGFR WT HEK293 cells (Fig. 4C, right). On the other hand, curcumin at the same dose did not exhibit any effects on EGFR expression (Fig. S1). To assess the inhibitory effects of 35d on EGFR signaling in the resistant cells, we reanalyzed our RNA-seq data. Gene set enrichment analysis (GSEA) analyses indicated that EGFR signaling down (DN) profiles were increased and EGFR signaling up profiles were decreased upon 35d treatment in GR6 cells (Fig. 4D). Thus, 35d reduced EGFR expression and comprehensively inhibited the EGFR signaling pathway in TKI-resistant cells. HSPA1B, encoding the hsp70, is a recognized molecular chaperone known to be involved in the targeting of oncogenic proteins for intracellular degradation (29Seo J. Han S.Y. Seong D. Han H.J. Song J. Multifaceted C-terminus of HSP70-interacting protein regulates tumorigenesis via protein quality control.Arch. Pharm. Res. 2019; 42: 63-75Crossref PubMed Scopus (14) Google Scholar). Our findings demonstrated that 35d upregulated HSPA1B gene expression and reduced EGFR protein expression. These data draw us to hypothesize that 35d may reduce EGFR expression by activating hsp70-mediated pathways. To test this, we first showed that hsp70 protein expression was substantially induced by 35d in GR6 and GR10 cells (Fig. 4, A and B right). Commercial hsp70 inhibitors pifithrin-μ (pif) and VER-155008 (ver) were then used to block hsp70-mediated pathways in EGFR-mutant, TKI-resistant cells. Inhibition of hsp70 by pif and ver reversed 35d-reduced EGFR expression in GR6 and GR10 cells (Fig. 4E), indicating that 35d led to suppression of EGFR protein through hsp70-mediated pathways. Hsp70 complex has been reported to involve lysosome or proteasome pathways as major degradation systems in the cells to regulate their client proteins (29Seo J. Han S.Y. Seong D. Han H.J. Song J. Multifaceted C-terminus of HSP70-interacting protein regulates tumorigenesis via protein quality control.Arch. Pharm. Res. 2019; 42: 63-75Crossref PubMed Scopus (14) Google Scholar, 30Chung C. Yoo G. Kim T. Lee D. Lee C.S. Cha H.R. et al.The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.Biochem. Biophys. Res. Commun. 2016; 479: 152-158Crossref PubMed Scopus (17) Google Scholar). Interestingly, our data showed that 35d reduced EGFR protein expression (Fig. 4A), but not mRNA expression (Fig. 4F), suggesting that 35d" @default.
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• W4376598246 title "Diarylheptanoid 35d overcomes EGFR TKI resistance by inducing hsp70-mediated lysosomal degradation of EGFR in EGFR-mutant lung adenocarcinoma" @default.
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