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PDF- 199K, A, Heterodimerization of integrinα2 or α5 with integrinβ1. B, Effects of integrinβ1, α2, or α5 siRNA, with or without erlotinib treatment, on downstream signaling molecules in PC9/ER2-2 cells.

PDF - 133K, A, PC9 and PC9/ER2-2 cells were exposed to 1 μM erlotinib with or without 0.1 μM dasatinib for 5 hr. B, The sensitivity to dasatinib after exposure for 3 days was determined by proliferation assay.

PDF - 82K, Supplementary materials and methods.

PDF - 163K, A, Establishment of stable integrinβ1-overexpressing cell lines (PC9/ITG2 and PC9/ITG11) or control cDNA transfected cell line (PC9/Mock) from PC9 cells by transfection with integrinβ1 cDNA. B, The effect of integrinβ1 overexpression on erlotinib sensitivity. Transfectants were treated with erlotinib for 3 days, and the IC50 values (μM) for erlotinib were 0.04 (PC9/Mock), 0.09 (PC9/ITG2), and 0.10 (PC9/ITG11). C, Western blots showing the expression and phosphorylation of EGFR, integrinβ1, Akt, and Erk1/2 following exposure to erlotinib for 5 hr.

PDF - 76K, Summary of expression states of integrins and resistance-related molecules in EGFR-TKI sensitive and refractory tumors.

PDF - 83K, Supplementary figure legends.

Highly effective organic ultraviolet (UV) filters, such as diethylamino hydroxybenzoyl hexyl benzoate (DHHB), are widely used in sunscreens; however, they have remarkably low solubility in water, which limits their use in water-based cosmetics. In this study, DHHB-encapsulated cationic polystyrene particles (PS-DHHB) dispersed in water were prepared via emulsion polymerization. Before polymerization, an emulsifying styrene monomer phase containing dissolved DHHB and a water phase containing low-molecular-weight poly(vinyl alcohol) were prepared, and subsequent polymerization yielded PS-DHHB particles. Crucially, the addition of polystyrene (PS) to the monomer phase contributed to a high solid content, and PS served as a stabilizer, enhancing the encapsulation efficiency of DHHB. Notably, the synthesized PS-DHHB exhibited UV absorption in a range similar to that of DHHB. Furthermore, even when aqueous alcohol (used as an antibacterial agent) was added, no DHHB was leached from the PS-DHHB particles. Finally, we found that the cationic PS-DHHB particles suppressed hair protein loss induced by UV light (320–400 nm) more effectively than cationic PS particles without DHHB. In summary, water-dispersed PS-DHHB particles exhibiting effective UV absorption and adsorption onto hair were developed, and these particles have applications in polymer science and cosmetics.

EGF receptor (EGFR) kinase inhibitors, including gefitinib and erlotinib, exert potent therapeutic efficacy in non–small cell lung cancers harboring EGFR-activating mutations. However, most patients ultimately develop resistance to these drugs. Here, we report a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors and the reversal of which could improve clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFR mutations that we established, there was increased expression of Src, integrin β1, α2, and α5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrin β1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Furthermore, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrin β1, α5, and/or α2 was also observed in refractory tumor samples from patients with lung cancer treated with erlotinib and/or gefitinib. Together, our findings identify the integrin β1/Src/Akt signaling pathway as a key mediator of acquired resistance to EGFR-targeted anticancer drugs. Cancer Res; 73(20); 6243–53. ©2013 AACR.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib have improved therapeutic efficacies on non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, appearance of drug resistant mutation limits patients' outcome. A secondary T790M mutation and MET amplification are two major mechanisms of acquired EGFR-TKI resistance. To develop novel therapeutics to overcome drug resistant tumors, we should further understand other mechanisms at molecular basis of intrinsic importance. We established erlotinib or gefitinib resistant cell lines from PC9 cells that harbor an activating EGFR deletion mutation by chronic exposure to these TKIs. We have previously identified that loss of PTEN or loss of activated EGFR gene allele is responsible for acquired resistance (Cancer Res., 2010; Nat Med., 2011; PLoS ONE, 2012; Cancer Sci., 2013). In our present study, one erlotinib-resistant subline from PC9 cells revealed following interesting characteristics; [1] The increased expression of integrins β1, α2, and α5 was observed, along with enhanced adhesion and migratory activity in the resistant sublines. [2] The increased expression and activation of Src was also observed. [3] Interestingly, integrinβ1 knockdown markedly restored sensitivity to erlotinib and reduced the activation of Src and Akt upon treatment with erlotinib. [4] Furthermore, Src knockdown suppressed Akt phosphorylation and cell growth, and this inhibitory effect was further augmented by erlotinib. [5] The expression of integrins β1 and α5 was also observed in a refractory tumor sample from a lung cancer patient treated with erlotinib. Together, we will present our novel concept that integrinβ1/Src/Akt pathway represents a novel a bypass effector pathway for the acquisition of resistance to EGFR-TKIs. Citation Format: Mayumi Ono, Rina Kanda, Kosuke Watari, Akihiko Kawahara, Masayoshi Kage, Hidetaka Uramoto, Michihiko Kuwano. The Integrinβ1/Src/Akt-driven bypass signaling pathway contributes to Erlotinib resistance in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2013-LB-220

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.