. . _:b51322660 . _:b604921144 . _:b51322661 . . _:b51322676 . . . _:b51322660 . _:b51322662 . _:b51322663 . _:b604921124 . . _:b51322677 "The levels of IGF-1 appear to correlate with cancer risk in human populations [>>27<<], and the high contents of IGF-1 are associated with increased mortality in the general population [28]." . _:b51322656 . _:b51322651 . . _:b51322657 . . _:b51322658 . . _:b604921129 . _:b604921152 . . _:b51322659 . . _:b51322668 . . . _:b51322669 . _:b604921111 "6"^^ . _:b51322670 . _:b51322671 . _:b51322671 . _:b51322664 . _:b604921139 . . _:b51322665 . _:b604921120 . . . _:b51322666 . . _:b51322667 . _:b604921170 . _:b51322676 . _:b604921117 "3"^^ . _:b51322677 . . _:b604921116 "3"^^ . _:b604921117 . _:b51322678 . _:b604921128 . . _:b604921119 "3"^^ . _:b51322679 . . . _:b604921118 "3"^^ . _:b51322652 "Crizotinib is a multitargeted TKI with activity against MET, ALK, and ROS1 [4, >>5<<]. This active substance has been found to greatly outperform the best chemotherapy agents in NSCLC patients with ALK rearrangements [6, 7]." . _:b51322672 . _:b51322656 . _:b604921113 "3"^^ . _:b604921126 . _:b51322673 . _:b604921149 . . _:b604921112 "4"^^ . _:b51322674 . _:b604921142 . _:b604921115 "3"^^ . . _:b51322675 . _:b604921114 "3"^^ . _:b604921125 "2"^^ . _:b604921158 . _:b604921124 "2"^^ . _:b604921150 . . . _:b604921127 "2"^^ . . _:b51322675 "Previously, we and other researchers reported that metformin in combination with EGFR-TKIs erlotinib or gefitinib had a synergistic inhibitory effect on the proliferation, migration, and invasion of cell lines resistant to TKIs [>>17<<, 26]. Taken together, data from the current study suggest that metformin, in combination with crizotinib, has the promising potential of turning into a novel anti-cancer agent that enhances the effect of crizotinib." . _:b51322662 . . _:b604921126 "2"^^ . . _:b51322680 . _:b604921121 "2"^^ . _:b51322660 "metformin in combination with gefitinib or erlotinib had a synergistic inhibitory effect on the proliferation, migration, and invasion of cell lines resistant to EGFR-TKIs (epidermal growth factor receptor-tyrosine kinase inhibitors) [>>17<<]. However, it remains unknown whether metformin may overcome crizotinib resistance." . _:b51322681 . _:b604921120 "2"^^ . _:b604921123 "2"^^ . . _:b604921122 "2"^^ . _:b51322656 . _:b51322665 "The relative cell invasion index was calculated as reported previously [>>17<<]. Triplicate samples of 2 \u00D7 104 cells treated as indicated were seeded into the upper well in serum-free medium and incubated with 10% FBS in the lower chambers." . _:b604921112 . _:b51322672 . _:b604921133 "2"^^ . _:b51322656 . _:b604921134 . . _:b604921132 "2"^^ . _:b604921141 . _:b51322646 "introduction" . _:b604921135 "2"^^ . . . _:b51322655 . _:b604921134 "2"^^ . _:b51322678 "The levels of IGF-1 appear to correlate with cancer risk in human populations [27], and the high contents of IGF-1 are associated with increased mortality in the general population [>>28<<]. Elevated IGF-1 concentrations were observed in patients who had taken crizotinib [29]. The amounts of IGF-1R were greater in tumor biopsies taken at the time of acquired resistance when compared to those obtained from the respective" . _:b604921136 . _:b604921137 . _:b604921129 "2"^^ . _:b604921138 . _:b51322668 "It was reported that metformin disrupted mitochondrial respiration, which then increased the intracellular ratio of AMP: ATP and induced activation of AMPK [>>20<<]. We thus analyzed the activation of AMPK caused by the metformin treatment. The results showed that metformin alone or in combination significantly stimulated the activation of AMPK (Figure 3). Taken together, these data suggest that" . _:b604921139 . _:b51322654 "This active substance has been found to greatly outperform the best chemotherapy agents in NSCLC patients with ALK rearrangements [6, >>7<<]. Recently, the PROFILE 1014 study demonstrated that crizotinib significantly improved PFS and ORR in the first-line treatment for ALK-positive NSCLC patients, with an acceptable safety profile, thus establishing crizotinib as a standard" . _:b604921140 . _:b604921141 . _:b604921128 "2"^^ . _:b604921142 . _:b604921143 . _:b604921144 . _:b604921145 . _:b51322657 "Gatekeeper mutations, such as L1196M, L1152R, C1156Y, and F1174L hinder drug binding and are frequently detected in crizotinib-resistant samples [>>13<<, 14]. Besides, the IGF-1R signaling pathway is involved in crizotinib resistance. In a previous study, the addition of IGF-1 induced resistance to the inhibitory effects of crizotinib on growth. Chronic ALK inhibition was associated with" . _:b604921131 "2"^^ . _:b604921146 . _:b604921147 . _:b604921167 . _:b604921114 . _:b604921148 . . _:b604921149 . _:b604921130 "2"^^ . . _:b604921150 . _:b51322668 . . _:b604921151 . _:b604921120 . . . _:b604921141 "2"^^ . _:b604921121 . . _:b604921169 . _:b604921122 . _:b604921123 . . _:b604921124 . _:b604921125 . _:b604921140 "2"^^ . _:b604921126 . _:b604921127 . _:b604921128 . _:b604921146 . _:b604921129 . _:b604921143 "2"^^ . _:b604921130 . _:b604921121 . _:b604921131 . _:b51322647 . _:b604921132 . _:b51322681 . _:b604921133 . _:b604921142 "2"^^ . . _:b604921134 . _:b604921135 . _:b604921137 "2"^^ . _:b51322681 "A synergistic in vitro growth inhibitory effect was established when an ALK inhibitor was combined with an mTOR inhibitor [>>32<<]. Thus, we investigated whether metformin overcomes crizotinib resistance through the inhibition of the IGF-1R signaling pathway." . _:b604921118 . _:b604921136 "2"^^ . _:b51322674 "In HER2-positive carcinomas, metformin in combination with the anti-HER2 monoclonal antibody trastuzumab could synergistically suppress the self-renewal and proliferation of cancer stem/progenitor cells [>>25<<]. Metformin is also involved in the targeted therapy of NSCLCs. Previously, we and other researchers reported that metformin in combination with EGFR-TKIs erlotinib or gefitinib had a synergistic inhibitory effect on the proliferation," . _:b604921111 . _:b604921112 . _:b604921111 . _:b604921113 . _:b604921139 "2"^^ . _:b604921114 . _:b51322670 "A combination of crizotinib with pemetrexed, or a targeted agent, such as an Hsp90 inhibitor, is currently being tested in clinical trials in ALK-positive advanced NSCLCs [>>7<<]. In the present study, we also adopted this approach, by using metformin in combination with crizotinib. As expected, the addition of metformin enhanced the sensitivity of H2228 and H3122 cells to crizotinib. Moreover, in the" . _:b604921115 . _:b604921116 . _:b604921117 . _:b604921151 . _:b604921138 "2"^^ . _:b604921118 . . _:b604921119 . . _:b604921149 "2"^^ . _:b51322663 _:b51322664 . _:b51322663 _:b51322665 . _:b51322663 _:b51322666 . _:b604921148 "2"^^ . _:b51322677 . _:b604921151 "2"^^ . _:b604921147 . _:b604921145 . _:b604921150 "2"^^ . . _:b604921117 . _:b604921145 "2"^^ . _:b604921156 . _:b51322669 _:b51322672 . _:b51322669 _:b51322673 . . _:b604921116 . _:b51322669 _:b51322674 . _:b51322669 _:b51322675 . _:b604921144 "2"^^ . _:b51322669 _:b51322676 . _:b51322667 _:b51322668 . _:b51322669 _:b51322677 . _:b604921119 . _:b51322669 _:b51322678 . _:b51322669 _:b51322679 . _:b51322648 "ALK rearrangements, first discovered by Soda et al. in 2007 [>>2<<], are either inversions or translocations of the ALK-tyrosine kinase (-TK) receptor gene with other fusion partners." . _:b604921147 "2"^^ . _:b51322669 _:b51322680 . _:b51322669 _:b51322681 . _:b604921118 . . _:b604921146 "2"^^ . . _:b604921153 . _:b604921113 . _:b604921157 "2"^^ . . . _:b51322679 . _:b51322664 . _:b604921112 . _:b604921156 "2"^^ . _:b604921115 . . _:b604921159 "2"^^ . _:b51322678 . _:b604921114 . _:b51322659 "Metformin has aroused keen interest as a potential anticancer agent ever since the report was published of the clinical evidence that the cancer risk and mortality were reduced in diabetics who received metformin [>>16<<]. Previously, we reported that metformin in combination with gefitinib or erlotinib had a synergistic inhibitory effect on the proliferation, migration, and invasion of cell lines resistant to EGFR-TKIs (epidermal growth factor" . _:b604921158 "2"^^ . _:b604921125 . _:b604921168 . _:b51322669 _:b51322670 . _:b604921125 . _:b604921169 . _:b604921153 "2"^^ . _:b51322669 _:b51322671 . _:b604921170 . _:b604921171 . _:b604921124 . _:b604921152 "2"^^ . _:b604921127 . _:b604921155 "2"^^ . . _:b604921126 . _:b604921154 "2"^^ . _:b604921152 . _:b51322666 "The detailed procedures of colony formation assay were described previously [>>34<<]. Briefly, 500 cells were resuspended in culture medium and seeded in six-well plates." . _:b604921121 . _:b604921153 . _:b51322650 "The product of the ALK rearrangement consists of a constitutively activated receptor TK with pro-oncogenic effects [>>3<<]. Consequently, tumors with ALK rearrangements are associated with ALK signaling, thus being effectively inhibited by small molecules ALK-TK inhibitors (-TKIs)." . _:b604921165 "2"^^ . _:b604921154 . _:b604921155 . _:b604921156 . _:b604921120 . _:b604921157 . _:b604921164 "2"^^ . _:b604921158 . _:b604921159 . _:b604921113 . _:b604921123 . _:b604921160 . _:b604921161 . _:b604921167 "2"^^ . _:b51322646 _:b51322660 . _:b604921157 . . _:b604921162 . _:b51322646 _:b51322661 . _:b604921163 . _:b51322646 _:b51322662 . _:b604921122 . _:b604921164 . _:b604921165 . _:b604921166 "2"^^ . _:b51322646 _:b51322656 . _:b604921166 . _:b51322646 _:b51322657 . _:b604921167 . . _:b51322646 _:b51322658 . _:b604921133 . _:b51322646 _:b51322659 . _:b604921161 "2"^^ . _:b51322646 _:b51322652 . _:b51322646 _:b51322653 . _:b51322646 _:b51322654 . . _:b604921132 . _:b51322646 _:b51322655 . _:b604921160 "2"^^ . _:b51322646 _:b51322648 . _:b604921140 . _:b51322646 _:b51322649 . _:b51322646 _:b51322650 . _:b604921135 . . _:b51322646 _:b51322651 . _:b604921163 "2"^^ . _:b604921143 . _:b604921123 . _:b604921134 . _:b51322646 _:b51322647 . _:b604921162 "2"^^ . _:b51322656 "The other mechanism is activation of alternative signaling pathways, including the development of EGFR mutations or activation of the wild-type EGFR, HER2-, or KIT-receptors [>>9<<\u201312]. Thus, innovative treatment strategies are urgently needed to overcome therapeutic resistance to crizotinib to improve the survival of NSCLC patients." . _:b604921129 . . _:b604921164 . _:b604921128 . _:b604921148 . _:b604921131 . _:b604921130 . _:b51322659 . . _:b604921141 . _:b604921169 "2"^^ . _:b604921140 . _:b604921168 "2"^^ . _:b604921159 . _:b604921143 . _:b604921171 "2"^^ . _:b51322680 "The activation of the mTOR pathway was related to increased autophagy of the ALK receptor, leading to a weakened response to crizotinib treatment [>>31<<]. A synergistic in vitro growth inhibitory effect was established when an ALK inhibitor was combined with an mTOR inhibitor [32]. Thus, we investigated whether metformin overcomes crizotinib resistance through the inhibition of the IGF-1R" . _:b604921142 . . _:b604921170 "2"^^ . _:b604921137 . _:b604921154 . _:b604921136 . _:b51322673 . . _:b604921139 . _:b51322663 "materials and methods" . . _:b604921138 . _:b604921160 . _:b604921149 . _:b51322653 "This active substance has been found to greatly outperform the best chemotherapy agents in NSCLC patients with ALK rearrangements [>>6<<, 7]. Recently, the PROFILE 1014 study demonstrated that crizotinib significantly improved PFS and ORR in the first-line treatment for ALK-positive NSCLC patients, with an acceptable safety profile, thus establishing crizotinib as a" . _:b604921148 . _:b51322658 . _:b604921136 . . "10.18632%2Foncotarget.9120" . _:b604921151 . _:b51322663 . _:b604921150 . _:b604921127 . . . _:b51322667 . _:b604921145 . _:b51322669 . . _:b604921144 . "PMC0" . _:b604921147 . _:b604921115 . . _:b604921146 . _:b51322649 . _:b604921157 . _:b51322658 "Gatekeeper mutations, such as L1196M, L1152R, C1156Y, and F1174L hinder drug binding and are frequently detected in crizotinib-resistant samples [13, >>14<<]. Besides, the IGF-1R signaling pathway is involved in crizotinib resistance. In a previous study, the addition of IGF-1 induced resistance to the inhibitory effects of crizotinib on growth. Chronic ALK inhibition was associated with" . _:b51322648 . . _:b604921156 . . _:b604921159 . _:b51322671 "In lung cancer patients with diabetes, meta-analysis showed that the use of metformin improved survival [>>22<<]. A retrospective study from our group showed that in advanced NSCLC patients with type 2 diabetes, metformin together with EGFR-TKI resulted in longer PFS and OS [23]. Metformin can target cancer stem cells, which are hypothesized to be" . . _:b51322651 "Crizotinib is a multitargeted TKI with activity against MET, ALK, and ROS1 [>>4<<, 5]. This active substance has been found to greatly outperform the best chemotherapy agents in NSCLC patients with ALK rearrangements [6, 7]." . _:b604921158 . _:b604921168 . _:b51322666 . _:b51322655 "significantly improved PFS and ORR in the first-line treatment for ALK-positive NSCLC patients, with an acceptable safety profile, thus establishing crizotinib as a standard of care for previously untreated ALK-positive patients [>>8<<]. However, acquired resistance eventually develops in most patients. Two main mechanisms of crizotinib resistance have been reported." . _:b604921153 . _:b604921152 . _:b604921155 . _:b604921138 . _:b51322646 . _:b51322652 . _:b604921154 . _:b604921165 . _:b604921164 . . _:b604921161 . . _:b604921167 . _:b604921166 . _:b51322661 . _:b604921161 . _:b51322647 "Genomic alterations in the anaplastic lymphoma kinase (ALK) gene are found in numerous malignancies [>>1<<]. ALK rearrangements, first discovered by Soda et al. in 2007 [2], are either inversions or translocations of the ALK-tyrosine kinase (-TK) receptor gene with other fusion partners." . _:b604921160 . _:b51322654 . . _:b604921163 . _:b604921132 . _:b604921162 . . . _:b604921131 . . _:b51322672 "A retrospective study from our group showed that in advanced NSCLC patients with type 2 diabetes, metformin together with EGFR-TKI resulted in longer PFS and OS [>>23<<]. Metformin can target cancer stem cells, which are hypothesized to be critical initiators of cancers [24]. In HER2-positive carcinomas, metformin in combination with the anti-HER2 monoclonal antibody trastuzumab could synergistically" . _:b604921116 . . _:b604921169 . _:b51322664 "Crizotinib-resistant cell lines were generated as previously reported [>>33<<]. Generally, parental cells were cultured with increasing concentrations of crizotinib." . _:b604921137 . _:b604921168 . _:b51322661 "Interestingly, metformin has been found to exert an inhibitory effect on IGF-1R signaling [>>18<<, 19], which encouraged us to perform the present study aimed at investigating whether metformin could restore crizotinib sensitivity in crizotinib-resistant cells through inhibition of the IGF-1R signaling pathway." . _:b604921171 . _:b51322667 "results" . _:b604921170 . _:b604921133 . _:b51322679 "The downstream signaling pathway, Akt/mTOR/S6 kinase pathway, was also associated with crizotinib resistance [>>30<<]. The activation of the mTOR pathway was related to increased autophagy of the ALK receptor, leading to a weakened response to crizotinib treatment [31]. A synergistic in vitro growth inhibitory effect was established when an ALK" . _:b51322676 "Previously, we and other researchers reported that metformin in combination with EGFR-TKIs erlotinib or gefitinib had a synergistic inhibitory effect on the proliferation, migration, and invasion of cell lines resistant to TKIs [17, >>26<<]. Taken together, data from the current study suggest that metformin, in combination with crizotinib, has the promising potential of turning into a novel anti-cancer agent that enhances the effect of crizotinib." . . . . _:b604921171 . . . . _:b51322670 . _:b604921111 . . . _:b604921166 . . . _:b604921165 . . _:b604921155 . . _:b51322680 . _:b51322675 . _:b51322662 "Interestingly, metformin has been found to exert an inhibitory effect on IGF-1R signaling [18, >>19<<], which encouraged us to perform the present study aimed at investigating whether metformin could restore crizotinib sensitivity in crizotinib-resistant cells through inhibition of the IGF-1R signaling pathway." . _:b51322669 "discussion" . _:b51322649 "The expression of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion oncogene, which is caused by a small inversion within chromosome 2p, was found in non-small cell lung cancer (NSCLC) [>>2<<]. The product of the ALK rearrangement consists of a constitutively activated receptor TK with pro-oncogenic effects [3]. Consequently, tumors with ALK rearrangements are associated with ALK signaling, thus being effectively inhibited by" . _:b51322673 "Metformin can target cancer stem cells, which are hypothesized to be critical initiators of cancers [>>24<<]. In HER2-positive carcinomas, metformin in combination with the anti-HER2 monoclonal antibody trastuzumab could synergistically suppress the self-renewal and proliferation of cancer stem/progenitor cells [25]. Metformin is also involved" . _:b51322646 . _:b51322647 . . . _:b604921130 . _:b51322665 . . _:b604921135 . _:b604921162 . . _:b51322653 . . _:b51322650 . _:b604921163 . _:b51322652 . _:b51322653 . . _:b604921122 . _:b51322654 . _:b604921119 . . . _:b51322655 . _:b51322648 . _:b51322674 . . _:b51322649 . . . _:b51322650 . _:b51322651 . . _:b51322657 .