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Killing of Kras-Mutant Colon Cancer Cells Via Rac- Independent Actin Remodeling by the Bgbp Cytokine, a Physiological PI3K Inhibitor Therapeutically Effective in Vivo

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Killing of Kras-Mutant Colon Cancer Cells Via Rac- Independent Actin Remodeling by the Bgbp Cytokine, a Physiological PI3K Inhibitor Therapeutically Effective in Vivo Published OnlineFirst July 2, 2012; DOI: 10.1158/1535-7163.MCT-11-1041-T Molecular Cancer Therapeutic Discovery Therapeutics Killing of Kras-Mutant Colon Cancer Cells via Rac- Independent Actin Remodeling by the bGBP Cytokine, a Physiological PI3K Inhibitor Therapeutically Effective In Vivo Livio Mallucci1, Dong-yun Shi1, Derek Davies2, Peter Jordan2, Alastair Nicol2, Lavinia Lotti4, Renato Mariani-Costantini5, Fabio Verginelli5, Valerie Wells3, and Daniel Zicha2 Abstract Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signaling pathways of which the most intensively studied are the Ras/ extracellular signal–regulated kinase cascade and the phosphoinositide 3-kinase (PI3K)/Akt cascade. How- ever, to date, there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here, we report that the b-galactoside–binding protein (bGBP) cytokine, a physiologic inhibitor of class I PI3Ks, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when coharboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis, which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis, and checkpoint kinase 2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice, we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-bGBP. Taken together, our results open a new therapeutic opportunity to a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu- r-bGBP may also be therapeutically effective against other cancers harboring activating Ras mutations as well as PIK3CA mutations. Mol Cancer Ther; 11(9); 1884–93. Ó2012 AACR. Introduction which controls cell proliferation and cell survival (8, 9) Activating mutations in the Ras genes (H,K,N; ref. 1), to be a major mediator of tumorigenesis. the most common oncogenic mutations in human cancers, The importance of activating mutations in the Ras are dominant determinants of drug resistance. Most fre- oncogenes in human cancers has made Ras and down- quent in Kras, Kras mutations define tumors refractory to stream effectors elective targets for therapeutic inter- conventional chemotherapy and radiation therapy (2, 3) vention, but the value of targeting inhibitors in the clinic and to more recently introduced therapies based on remains doubtful. For example, prevention of Ras anchor- EGF receptor tyrosine kinase inhibitors (4–7). Locked in age at the cell membrane by farnesyltransferase inhibi- the GTP-bound mode, oncogenic Ras constitutively acti- tors has been shown to be effective in mice expressing vates the Raf/MEK/extracellular signal–regulated kinase oncogenic Ras but not in patients with solid tumors (ERK) cascade and an integrated signaling network, (10). Similarly, in more recent studies, CI-1040, a MEK inhibitor, was found to induce significant shrinkage of Kras-driven lung carcinomas in mice (11), but had no fi 1 Authors' Af liations: School of Biomedical and Health Sciences, King's significant effect in patients with advanced lung, breast, College London; 2Cancer Research UK London Research Institute; 3NYU in London, London, United Kingdom; 4Department of Experimental Medicine, colon, and pancreatic cancers (12). Using a different La Sapienza University, Rome; and 5Unit of General Pathology, Aging approach, a number of compounds with efficacy against Research Center, G. D'Annunzio University Foundation, Chieti, Italy cells with mutant-activated Kras have been identified by Note: Current address for D.-y. Shi: Department of Biochemistry and high-throughput screening (13–16) and shown to inhibit Molecular Biology, Shanghai Medical College of Fudan University, 200032 Shanghai, PR China. xenograft growth (13, 15, 16); however, the mechanisms by which these compounds operate are not fully under- Corresponding Author: Livio Mallucci, School of Biomedical and Health Sciences, King's College London, Franklin Wilkins Building, 150 Stamford stood and their anticancer efficacy in the clinic remains Street, SE1 9NH London, United Kingdom. Phone: 44-207-848-4257; Fax: uncertain. 44-207-848-4500; E-mail: [email protected] Recently, the necessary role played by the phosphoi- doi: 10.1158/1535-7163.MCT-11-1041-T nositide 3-kinase (PI3K)/Akt pathway in maintaining Ó2012 American Association for Cancer Research. tumor growth has made this pathway a target for small 1884 Mol Cancer Ther; 11(9) September 2012 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst July 2, 2012; DOI: 10.1158/1535-7163.MCT-11-1041-T Killing Kras-Mutant Cancer Cells A SW480 SW620 β LoVo β βGBP GBP GBP -5 β β β 60 Control GBP nmol/L 120 Control GBP nmol/L 80 Control GBP nmol/L 50 0 100 0 0 60 Figure 1. Treatment with 40 80 30 2 Hu-r-bGBP inhibits cell proliferation Day 3 1 60 Day 3 40 Day 3 20 40 and induces apoptosis. A, rate of cell 1 20 10 2 20 proliferation as related to dose 2 4 4 4 Cell number × 10 0 00 response. Values are means of 0001 2333444555 1122 triplicate cultures Æ SEM. Insets Day Day Day show cell-cycle phase distribution at day 3 of treatment with 4 nmol/L Hu- B Control βGBP Control βGBP Control βGBP b b 5 5 5 5 r- GBP. Hu-r- GBP was added 6 105 10 10 10 105 10 hours after seeding. B, apoptotic 1.9 9.2 2.2 38.8 4.7 25.0 events at day 3 of treatment. Top to 103 103 103 103 103 103 DAPI DAPI DAPI bottom, mitochondrial membrane 101 101 101 101 101 101 potential assessed by 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 tetramethylrhodamine ester (TMRE) TMRE TMRE TMRE staining; phosphatidylserine orientation at the plasma membrane 105 105 105 105 105 105 assessed by Annexin V staining; 1.7 4.6 2.2 23.6 1.3 13.6 103 103 103 103 103 103 DAPI DAPI caspase-3 activity assessed using DAPI PhiPhiLux. Inset values are 101 101 101 101 101 101 percentages of cells committed to 101 103 105 101 103 105 101 103 105 101 103 105 101 103 105 101 103 105 apoptosis (encircled area). Hu-r- Annexin Annexin Annexin bGBP 4 nmol/L added 6 hours after 5 5 5 5 5 seeding. Data are from parallel 10 10 105 10 10 10 representative experiments. 1.0 4.5 0.9 23.9 0.4 8.3 103 103 103 103 103 103 DAPI DAPI DAPI 101 101 101 101 101 101 101 103 105 101 103 105 101 103 105 101 103 105 101 103 105 101 103 105 Caspase-3 Caspase-3 Caspase-3 K Â À10 molecule inhibitors of the PI3K cascade (17–21). However, binding ( d 1.5 10 mol/L; ref. 26) and molecular the efficacy of these inhibitors as single agents in Kras- interactions leading to downregulation of class IA and driven oncogenesis is disputable. For example, in mice class IB PI3Ks (27). Inhibition of PI3K activity by Hu-r- that had developed lung tumors in response to Kras bGBP was found to have two major outcomes: sup- activation, no significant tumor regression was found pression of Ras-GTP loading, leading to a block of ERK upon treatment with a dual pan-PI3K/mTOR inhibitor, activation (27) and negation of Akt gene expression which had instead shown efficacy in lung tumors driven leading to loss of Akt (21), conditions that either by by the expression of activated PI3K (22). In other experi- blocking the ability of cancer cells to proliferate or by ments, the efficacy of the PI3K inhibitor PX-866 was impairing their ability to survive can block oncogenic- significantly less in xenografts bearing Kras and PIK3CA ity, thus highlighting a selective anticancer effect as in mutations than in those having PIK3CA mutations alone normal cells, bGBP-enforced cell cycle restriction is (23). On the other hand, Kras-mutant lung tumors did reversible (26). regress when the pan-PI3K/mTOR inhibitor was used in We now report that in colorectal carcinoma cells bearing combination with an MEK inhibitor (22). Similarly, in cells oncogenic Kras downregulation of PI3K activity by Hu-r- expressing Kras or Hras mutants, resistance to PI3K bGBP at doses that did not inhibit canonical signaling inhibitors could be reversed by inhibitors of the Ras/ERK downstream of Ras or PI3K promoted events critical to the pathway (24). Despite these positive results, it remains to activation of an intrinsic and an extrinsic cell death pro- be established whether in the human system Kras onco- gram initiating with Rac-independent actin reorganiza- genicity could be overcome by the combined targeting of tion assignable to phosphoinositide changes at the plasma the PI3K and the Ras pathways and be beneficial as, in membrane. We also report that Hu-r-bGBP had therapeu- addition to toxicity, a disadvantage of this approach is that tic efficacy in vivo and that the added presence of activat- these pathways are also required for the proliferation of ing mutations in PIK3CA did not confer resistance to normal cells and for the maintenance of their homeostatic treatment with Hu-r-bGBP.
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