Cytostatic Response Research Articles

Galectin-1 Controls the Proliferation and Migration of Liver Sinusoidal Endothelial Cells and Their Interaction With Hepatocarcinoma Cells.

Galectin-1 (Gal1), a β-galactoside-binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial-mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor β1 (TGF-β1 ) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF-β1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF-β1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH-7 human HCC cells, and in SK-HEP-1 human liver sinusoidal endothelial cells (SECs). We used loss-of-function and gain-of-function experiments to down- or up-regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK-HEP-1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch-wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan-dependent heterotypic adhesion of HepG2 cells to SK-HEP-1 SECs. Furthermore, TGF-β1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK-HEP-1 SECs through a Gal1-dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF-β1 -induced growth inhibition. Our results suggest that Gal1 and TGF-β1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis.

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β.

Loss of a cytostatic response to TGF-β has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-β have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-β may open up new avenues for the development of therapeutic strategies. Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-β on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. We found that the OPN expression levels dose-dependently decreased in TGF-β-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-β-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-β inhibitory element found in other TGF-β-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-β-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-β occurred concomitantly with loss of the TGF-β anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-β signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-β.

Abstract B03: Pin1 is required for sustained B cell proliferation upon oncogenic activation of Myc

Abstract The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma, where it is also frequently subject to secondary mutations. Substitutions in codon 58, in particular, stabilize the Myc protein and augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 primes phosphorylation of Thr 58, providing a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, genetic data are missing to address whether Pin1 influences Myc-induced lymphomagenesis. Here, we show that genetic ablation of Pin1 in Eµ-myc transgenic mice starkly reduced lymphoma onset and penetrance. In pre-malignant Pin1 deficient B cells, the proliferative response to Myc was selectively impaired, while Myc-induced apoptosis was intact. This phenotype was not the result of deregulated levels of Ser 62 phosphorylation in the Pin1-/- background. Similar results were observed in Pin1-/- mouse embryo fibroblasts (MEFs), where prolonged Myc activation inhibited proliferation. In either B cells or MEFs, loss of Pin1 did not affect mitogen-induced cell cycle entry, an effect that relies on endogenous Myc activity. Thus, our data unravel the existence of a synthetic-lethal interaction between deletion of Pin1 and oncogenic activation of Myc. Myc-induced oncogenic stress is linked to the induction of the Arf/p53 pathway, which is critical for tumor suppression in Eµ-myc mice. The selective pressure to inactivate Arf/p53 axis was preserved in Eµ-myc Pin1-/- lymphomas. In the absence of Arf or p53, the Pin1 deletion no longer reduced Myc-induced tumorigenesis or proliferation. Thus the proliferative arrest of pre-tumoral Eµ-myc Pin1-/- B cells required an active ARF-p53 dependent cytostatic response. Our data demonstrate that Pin1 is required to support the oncogenic activity of Myc in B cells and warrant further assessment of Pin1 as a therapeutic target in Myc-dependent malignancies. Citation Format: Luana D'Artista, Andrea Piontini, Theresia Kress, Marco Morelli, Alessandro Verrecchia, Mirko Doni, Stefano Campaner, Bruno Amati. Pin1 is required for sustained B cell proliferation upon oncogenic activation of Myc. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B03.

The Effects of miR-20a on p21: Two Mechanisms Blocking Growth Arrest in TGF-β-Responsive Colon Carcinoma.

Loss of response to TGF-β is a central event in the genesis of colorectal cancer (CRC), a disease that, in the majority cases, is refractory to growth inhibition induced by this cytokine. However, inactivating mutations at receptors and transducers from the TGF-β cascade occur only in approximately half of CRCs, suggesting the involvement of additional mechanisms altering the response to the cytokine. We have recently described the amplification of the 13q31 locus, where the miR-17-92 cluster maps, associated with overexpression of its members. In this study, we address the potential role of miR-20a, from the miR-17-92 cluster, in the suppression of TGF-β cytostatic response in CRC. Using the poorly tumorigenic and TGF-β-sensitive FET cell line that expresses low miR-20a levels, we first confirmed that miR-20a downmodulated CDKN1A expression, both at mRNA and protein level, through direct binding to its 3'-UTR. We demonstrated that miR-20a significantly diminished cell response to TGF-β by preventing its delay of G1/S transition and promoting progression into cell cycle. Moreover, besides modulating CDKN1A, miR-20a blocked TGF-β-induced transactivation of its promoter without affecting the post-receptor activation of Smad3/4 effectors directly. Finally, miR-20a abrogated the TGF-β-mediated c-Myc repression, a direct inhibitor of the CDKN1A promoter activation, most likely by reducing the expression of specific MYC-regulating genes from the Smad/E2F-based core repressor complex. Our experiments indicate that miR-20a interferes with the colonic epithelium homeostasis by disrupting the regulation of Myc/p21 by TGF-β, which is essential for its malignant transformation.

Abstract 1843: Novel actions of next-generation taxanes benefit advanced stages of prostate cancer

Abstract Purpose: To improve the outcomes of patients with castrate resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individual specific treatments for patients with CRPC. The current studies compared the novel taxane, cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond. Experimental design: Cabazitaxel (CBTX) and docetaxel (DCTX) were compared via in vitro modeling to determine molecular mechanism, biochemical and cell biological impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro, and in xenograft tumors for cabazitaxel response. Results: The data herein show that i. CBTX exerts stronger cytostatic and cytotoxic response compared to DCTX, especially in CRPC; ii. CBTX induces aberrant mitosis, leading to pyknotic and multinucleated cells; iii. taxanes do not act through the androgen receptor (AR); iv. Gene expression profiling reveals distinct molecular actions for CBTX v. tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to CBTX. Conclusions: CBTX not only induces improved cytostatic and cytotoxic effects, but also impacts distinct molecular pathways, compared to DCTX, which could underlie its efficacy after DCTX treatment has failed in CRPC patients. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization taxanes, which could benefit up to 50% of CRPC cases. Citation Format: Renee de Leeuw, Lisa Berman-Booty, Matthew Schiewer, Stephen Ciment, Robert Den, Adam Dicker, William Kelly, Edouard Trabulsi, Costas Lallas, Leonard Gomella, Karen Knudsen. Novel actions of next-generation taxanes benefit advanced stages of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1843. doi:10.1158/1538-7445.AM2015-1843

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5'-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

Novel actions of next-generation taxanes benefit advanced stages of prostate cancer.

To improve the outcomes of patients with castration-resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individualize specific treatments for patients with CRPC. These studies compared the novel taxane cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond. Cabazitaxel and docetaxel were compared via in vitro modeling to determine the molecular mechanism, biochemical and cell biologic impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro and in xenograft tumors for cabazitaxel response. The data herein show that (i) cabazitaxel exerts stronger cytostatic and cytotoxic response compared with docetaxel, especially in CRPC; (ii) cabazitaxel induces aberrant mitosis, leading to pyknotic and multinucleated cells; (iii) taxanes do not act through the androgen receptor (AR); (iv) gene-expression profiling reveals distinct molecular actions for cabazitaxel; and (v) tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to cabazitaxel. Cabazitaxel not only induces improved cytostatic and cytotoxic effects, but also affects distinct molecular pathways, compared with docetaxel, which could underlie its efficacy after docetaxel treatment has failed in patients with CRPC. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization to taxanes, which could benefit up to 50% of CRPC cases.

Game of isoforms: PI3K β-sparing inhibitor is coming.

Cutaneous melanoma has been at the forefront of targeted therapy breakthroughs over the last 5 years. The FDA approval of BRAF and MEK inhibitors for the treatment of late-stage, mutant V600E/K BRAF-harboring melanoma patients has been heralded due to high response rates and improvements in progression-free survival, but the durable effects of these inhibitors are limited. Alterations in the phosphoinositide 3-kinase (PI3K) pathway including inactivation of PTEN (a tumor suppressor that normally down-regulates PI3K signaling) and, less frequently, mutations in PIK3CA (the gene encoding the α isoform of the PI3K catalytic subunit) have been identified in mutant BRAF melanomas. Furthermore, PI3K pathway signaling is often up-regulated in association with resistance to mutant BRAF and/or MEK targeting agents. Despite the evidence highlighting the importance of the PI3K pathway in melanoma, initial testing of class I PI3K inhibitors in patients has not produced dramatic results. An issue for combinatorial studies is the overlapping toxicities of MAPK pathway inhibitors and PI3K-targeting agents that limits their effective dosing. A recent study from Deuker and colleagues provides important preclinical advances in this area by examining the PI3K isoform dependencies in mutant BRAF melanomas. PI3K functions as a heterodimer with a p110 catalytic subunit and a regulatory subunit. Four isoforms of p110 subunits exist: α, β, δ and γ (Jean and Kiger, 2014; Yuan and Cantley, 2008). p110α and p110β are ubiquitously expressed, whereas δ and γ isoforms display highest expression in immune cells. Importantly, isoform-selective PI3K inhibitors have been developed to target tumor dependencies and minimize toxicities in non-tumor cells. Deuker et al. utilized these selective inhibitors to determine the PI3K isoform dependency of mutationally active PI3Kα and PTEN-deficient subgroups of mutant BRAF melanomas using cell lines derived from mouse models and human patients. In vitro, PTEN-deficient lines were less sensitive to the α-isoform PI3K inhibitor, BYL719, compared to mutant H1047R PI3Kα cells. In contrast to other cancer types, in which PTEN-deficient tumors are often PI3Kβ-dependent (Wee et al., 2008), PTEN-null melanomas did not rely on PI3Kβ. Growth of PTEN-null mutant BRAF melanomas was blocked by a PI3Kα/δ/γ-targeting but β-sparing inhibitor, GDC-0032. In addition, the δ/γ inhibitor, IPI145, enhanced the effects of BYL719 suggesting involvement of the δ/γ isoforms in melanoma. PI3Kα-dependency is also suggested to be present in BRAF V600E/WT PTEN melanomas, which represent a substantial proportion of mutant BRAF melanomas, although confirmation is warranted in a larger panel of BRAF V600E/WT PTEN melanoma models. “More dramatic tumor regressions were observed in vivo with the combination of BRAF-MEK pathway and PI3K inhibitors. ” A long-term limitation to both BRAF and MEK inhibitors in melanoma is acquired drug resistance and patients who develop resistance have few second-line treatment options. In addition to up-front combination, treatment of MEK inhibitor-resistant tumors with the PI3K β-sparing inhibitor resulted in a cytostatic response. These data were generated from a single MEK inhibitor-resistant tumor. As multiple mechanisms of resistance are likely to occur, it will be important to further explore effects in cultured- and patient-derived BRAF and MEK inhibitor-resistant models. Overall, the study presented by Deuker et al., provides an important preclinical advance in targeting the PI3K pathway in both first-line combinations as well as a possible second-line salvage option for BRAF-MEK pathway resistant melanomas. These findings should propel investigation of methods to quantitatively and non-invasively measure PI3K signaling in preclinical in vivo melanoma models and to determine the most effective dosing regimens. Given the high degree of immune reactivity of melanomas and remarkable advances in immune-based therapies, other key issues will be to determine the effect of targeting PI3K on infiltration of immune cells into melanomas and the extent to which PI3K-targeting agents can be combined with immune-based therapies. Advances in mutant BRAF melanomas will lead to studies addressing the importance of PI3K isoform specific targeting in wild-type BRAF subsets of melanoma for which there is an unmet need for new therapeutic strategies.

High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response.

Therapeutic targeting of cellular metabolism in cells with hyperactive mTORC1: a paradigm shift.

mTORC1 is an established master regulator of cellular metabolic homeostasis, via multiple mechanisms that include altered glucose and glutamine metabolism, and decreased autophagy. mTORC1 is hyperactive in the human disease tuberous sclerosis complex (TSC), an autosomal dominant disorder caused by germline mutations in the TSC1 or TSC2 gene. In TSC-deficient cells, metabolic wiring is extensively disrupted and rerouted as a consequence of mTORC1 hyperactivation, leading to multiple vulnerabilities, including "addiction" to glutamine, glucose, and autophagy. There is synergy between two rapidly evolving trajectories: elucidating the metabolic vulnerabilities of TSC-associated tumor cells, and the development of therapeutic agents that selectively target cancer-associated metabolic defects. The current review focuses on recent work supporting the targeting of cellular metabolic dysregulation for the treatment of tumors in TSC, with relevance to the many other human neoplasms with mTORC1 hyperactivation. These data expose a fundamental paradox in the therapeutic targeting of tumor cells with hyperactive mTORC1: inhibition of mTORC1 may not represent the optimal therapeutic strategy. Inhibiting mTORC1 "fixes" the metabolic vulnerabilities, results in a cytostatic response, and closes the door to metabolic targeting. In contrast, leaving mTORC1 active allows the metabolic vulnerabilities to be targeted with the potential for a cytocidal cellular response. The insights provided here suggest that therapeutic strategies for TSC and other tumors with activation of mTORC1 are at the verge of a major paradigm shift, in which optimal clinical responses will be accomplished by targeting mTORC1-associated metabolic vulnerabilities without inhibiting mTORC1 itself.

Abstract 4295: A 3D culture model for screening of cancer therapeutics

Abstract 3D cell culture models are currently of great interest to the bioscience community. This is most notable in the cancer biology area, where a growing body of evidence suggests that more relevant and translational observations can be made by testing compounds in a physiological relevant 3D model compared to a 2D model. In this study, we take a kinetic, live-cell imaging approach to measure the growth or shrinkage of non-adherent tumor spheroids using Ultra Low Attachment (ULA) 96-well and 384-well microtiter plates (Corning®). These round-bottomed, hydrogel coated plates facilitate the formation of spheroids by promoting self-adherence as opposed to adherence to the plate surface. In a single cell type, monoculture model, A549 lung epithelial carcinoma cells developed into single spheroid structures in each well of 96 and 384-well plates within 48-96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 µm - 600 µm, was proportional to the number of cells (1K - 5K cells per well). Post formation, spheroids remained untreated or were subjected to three test agents, staurosporine, oxaliplatin or cycloheximide. The increase or decrease in spheroid size was monitored over 3 - 10 days using the IncuCyte™ ZOOM Live Content Imaging system. Using the red fluorescent channel, spheroid growth was analyzed using fluorescent area and intensity metrics. Whereas staurosporine and oxaliplatin significantly decreased spheroid size at high concentrations, cycloheximide treatment resulted in a cytostatic response, effectively inhibiting spheroid growth in a concentration dependent manner, while not reducing total spheroid area at any tested concentration. Interestingly, in side-by-side comparisons of kinetic 2D and 3D assays, we observed a significant, 10-fold (one log) shift in the pharmacology of cycloheximide. In a 3D co-culture breast cancer model, this study also illustrates the effect of Lapatinib on SK-BR-3 cells grown in the presence of mammary derived stromal cells (fibroblasts). Together, this study describes a kinetic, live-cell approach to measuring spheroid size over time, illustrates a differential response of cycloheximide in a 3D system compared to identically treated cells grown in a 2D monolayer, and details a complex co-culture method for elucidating the effect of Lapatinib in a physiologically relevant 3D model of breast cancer. Citation Format: Kalpana Patel, Belinda O'Clair, Tim O'Callaghan, Daniel M. Appledorn, Derek Trezise. A 3D culture model for screening of cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2014-4295

Abstract 2925: Mechanism-based combinatorial treatment of KRAS mutant colorectal cancer with MEK and PI3K pathway inhibitors

Abstract Treatment of metastatic colorectal cancer remains a considerable hurdle as typically <20% of patients respond to available targeted drugs. The most advanced treatment options for these patients are anti-EGFR therapies. However, it is now accepted that tumors with KRAS mutations will not respond to these therapies. Genomic aberrations, including KRAS mutation, frequently activate the MAPK and PI3K pathways in cancer, and combined inhibition of these pathways are now being trialed widely in the clinic. We used a panel of 28 colorectal cancer cell lines to explore combinations of PI3K (PI3Ki) and MEK inhibitors (MEKi). As expected BRAF mutant tumors were sensitive to allosteric MEKi. KRAS/BRAF mutant tumors were resistant to PI3K pathway inhibitors compared to wild-type cells, while tumors with PIK3CA and KRAS/BRAF mutations had intermediate sensitivity. PI3Ki induced a robust G1 cell cycle arrest in sensitive cells and a potent autophagic response in less sensitive lines. MEKi induced a cytostatic response. Combination of MEKi with PI3K pathway inhibitors of different chemotype and selectivity demonstrated that class I PI3Ki were potent inducers of synergy with MEK inhibition. Combination of the MEKi with mTOR inhibitors was at best additive, while class I PI3K/mTOR inhibitors were borderline synergistic/additive with MEKi. Normal non-transformed colon cell lines did not show synergy, which was in part due to a lack of response to the MEKi. We selected a combination of GDC0941 (class I PI3Ki) with GDC0973 (MEKi) to explore the mechanism behind the synergistic interaction. A potent combinatorial effect was observed at the level of FOXO-regulated transcription. Regulators of apoptosis were affected both at the transcriptional and post-translational level by the combination, and this resulted in switching from cytostasis/autophagy to an apoptotic response. Gene knockout and a siRNA screen targeting apoptotic regulators identified a dual requirement for BIM and BBC3 BH3-proteins that acted through activation of BAX and abrogation of MCL1/BCL-XL to drive apoptosis. Continuous exposure to the combination resulted in acquired resistance, through resistance to the MEKi. This resistant line retained sensitivity to an ERK inhibitor and a synergistic interaction when combined with GDC0941. Overall, our data suggest that combination of MEK and class I PI3Ki may be a treatment option for the particularly hard to treat KRAS mutant colorectal cancers. Resistance to targeted agents is major challenge in clinic and may be overcome by combinatorial treatments. Intriguingly, our observations indicate that, as with single agent treatments, cancer cells can also evolve resistance to combinatorial regimes. This suggests that combined treatment with two targeted agents may not be sufficient to overcome resistance and that combinations of multiple (>2) agents or intermittent/alternating treatment strategies will be required. Citation Format: Paul A. Clarke, Toby Roe, Paul Workman. Mechanism-based combinatorial treatment of KRAS mutant colorectal cancer with MEK and PI3K pathway inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2925. doi:10.1158/1538-7445.AM2014-2925

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence.

Anticancer and antioxidant activity of bread enriched with broccoli sprouts.

This study is focused on antioxidant and anticancer capacity of bread enriched with broccoli sprouts (BS) in the light of their potential bioaccessibility and bioavailability. Generally, bread supplementation elevated antioxidant potential of product (both nonenzymatic and enzymatic antioxidant capacities); however, the increase was not correlated with the percent of BS. A replacement up to 2% of BS gives satisfactory overall consumers acceptability and desirable elevation of antioxidant potential. High activity was especially found for extracts obtained after simulated digestion, which allows assuming their protective effect for upper gastrointestinal tract; thus, the anticancer activity against human stomach cancer cells (AGS) was evaluated. A prominent cytostatic response paralleled by the inhibition of AGS motility in the presence of potentially mastication-extractable phytochemicals indicates that phenolic compounds of BS retain their biological activity in bread. Importantly, the efficient phenolics concentration was about 12 μM for buffer extract, 13 μM for extracts after digestion in vitro, and 7 μM for extract after absorption in vitro. Our data confirm chemopreventive potential of bread enriched with BS and indicate that BS comprise valuable food supplement for stomach cancer chemoprevention.

A Case Series Of Continuous Low Dose Lenalidomide In Patients With Relapsed Or Refractory Classical Hodgkin Lymphoma

BackgroundRelapsed or refractory (rel/ref) classical Hodgkin lymphoma (cHL) remains a clinical challenge with limited effective treatments after high dose therapy with autologous stem cell transplantation (ASCT). Lenalidomide (Revlimid®) is an approved anti-neoplastic therapy for multiple myeloma, mantle cell lymphoma and myelodysplastic syndrome with del(5q). It has emerged as an agent with a manageable toxicity profile and promising clinical activity in a number of B cell malignancies. Two clinical trials have evaluated lenalidomide in rel/ref patients with cHL at a dose of 25mg daily for 21 days of a 28 day cycle. One study reported an overall response rate of 14%, a median time to progression of 3.2 month, with a median number of prior therapies of 2. The second study reported an overall response rate of 19%, a cytostatic response rate (CR+PR+SD> 6 month) of 33%, with a median number of prior therapies of 4. The most common reported grade 3-4 side effects in these studies respectively were: neutropenia 28% and 47%, thrombocytopenia 28% and 27%, and anemia 21% and 27%. We report on our experience in this case series with continuous low dose (10-20mg) lenalidomide in rel/ref cHL patients. MethodsTwelve rel/ref cHL patients (pts) who previously underwent (or were not candidates for) ASCT and/or clinical trials were administered a daily dose of lenalidomide. Pts received 10mg and were titrated up to 20mg, if tolerated, with continuous dosing for 30 day cycles. Treatment continued until disease progression or the development of unacceptable adverse event at the lowest administered dose (5mg) of lenalidomide. ResultsThe median age at treatment was 33 (range 24-61) years with 5 females. Median number of prior therapies was 8 (range 3-16). Ten pts had received a prior stem cell transplant (9 ASCT, 1 allogenic, and 1 both ASCT and allogeneic). Of the 12 pts, we observed 3 PR (25%), and 9 SD (8 for more than 6 months) for an overall response rate of 25% and an overall cytostatic response rate (CR+PR+SD> 6 month) of 92%. Median number of cycles received was 6 (range 2-15); six patients remain on therapy. One patient with a PR after 2 cycles of therapy underwent allogeneic stem cell transplant. The median time to progression for the remaining 11 patients was 7.5 months (range 4-15 months). Three patients had progression of disease, all of whom were able to enroll on clinical trials. One patient discontinued therapy because of exacerbation of preexisting neuropathy and was able to transition to chemotherapy. A second patient discontinued therapy after experiencing significant fatigue. In general, the treatment was well tolerated, and the most common clinically significant adverse events were: neutropenia in 4 patients which was managed by GCSF support and dose reduction, exacerbation of neuropathy in 2 patients with one patient discontinuing therapy and dose reduction in the second, and diarrhea in one patient managed with dose reduction. ConclusionsDespite a far more heavily treated patient population, continuous, low dose, single agent lenalidomide was both tolerable and effective in patients with rel/ref cHL. Continuous low dose lenalidomide is able to provide meaningful time to progression and bridge to further therapy. A clinical trial to evaluate the efficacy of continuous daily dosing of lenalidomide in rel/ref cHL is warranted. Disclosures:Off Label Use: Lenalidomide in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma. O'Connor:Celgene: Consultancy, Research Funding.

Triterpene saponosides from Lysimachia ciliata differentially attenuate invasive potential of prostate cancer cells

Neither androgen ablation nor chemotherapeutic agents are effective in reducing the risk of prostate cancer progression. On the other hand, multifaceted effects of phytochemicals, such as triterpene saponins, on cancer cells have been suggested. A promising safety and tolerability profile indicate their possible application in the treatment of advanced prostate cancers. We analyzed the specificity, selectivity and versatility of desglucoanagalloside B effects on human prostate cancer cells derived from prostate cancer metastases to brain (DU-145 cells) and bone (PC-3 cells). Prominent growth arrest and apoptotic response of both cell types was observed in the presence of sub-micromolar desglucoanagalloside B concentrations. This was accompanied by cytochrome c release and caspase 3/7 activation. A relatively low cytostatic and pro-apoptotic response of cancer cells to a desglucoanagalloside B analog, anagallosaponin IV, illustrated the specificity of the effects of desglucoanagalloside B, whereas the low sensitivity of normal prostate PNT2 cells to desglucoanagalloside B showed the selectivity of its action. Inhibition of cancer cell motility was observed in the presence of both saponins, however only desglucoanagalloside B attenuated cancer cell invasive potential, predominantly through an effect on cell elastic properties. These data demonstrate the versatility of its effects on prostate cancer cells. In contrast to PNT2 cells, cancer cells tested in this study were relatively resistant to mitoxantrone. The multifaceted action of desglucoanagalloside B on basic cellular traits, crucial for prostate cancer progression, opens perspectives for elaboration of combined palliative therapies and new prostate cancer prophylaxis regimens.

That which does not kill me makes me stronger; combining ERK1/2 pathway inhibitors and BH3 mimetics to kill tumour cells and prevent acquired resistance

Oncogenic mutations in RAS or BRAF can drive the inappropriate activation of the ERK1/2. In many cases, tumour cells adapt to become addicted to this deregulated ERK1/2 signalling for their proliferation, providing a therapeutic window for tumour-selective growth inhibition. As a result, inhibition of ERK1/2 signalling by BRAF or MEK1/2 inhibitors is an attractive therapeutic strategy. Indeed, the first BRAF inhibitor, vemurafenib, has now been approved for clinical use, while clinical evaluation of MEK1/2 inhibitors is at an advanced stage. Despite this progress, it is apparent that tumour cells adapt quickly to these new targeted agents so that tumours with acquired resistance can emerge within 6-9 months of primary treatment. One of the major reasons for this is that tumour cells typically respond to BRAF or MEK1/2 inhibitors by undergoing a G1 cell cycle arrest rather than dying. Indeed, although inhibition of ERK1/2 invariably increases the expression of pro-apoptotic BCL2 family proteins, tumour cells undergo minimal apoptosis. This cytostatic response may simply provide the cell with the opportunity to adapt and acquire resistance. Here we discuss recent studies that demonstrate that combination of BRAF or MEK1/2 inhibitors with inhibitors of pro-survival BCL2 proteins is synthetic lethal for ERK1/2-addicted tumour cells. This combination effectively transforms the cytostatic response of BRAF and MEK1/2 inhibitors into a striking apoptotic cell death response. This not only augments the primary efficacy of BRAF and MEK1/2 inhibitors but delays the onset of acquired resistance to these agents, validating their combination in the clinic. This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects

We report a real-time cell analysis (RTCA) sensing method of 96 electronic microwells for profiling the cytotoxicity of nanoparticles on different cell lines. The method consists of 96 microwells embedded with microelectrodes (96x E-plate) to measure impedance changes of adherent cell lines. When the testing cells change in population, adhesion, and/or morphology, the impedance at the cell–electrode interface changes to provide real-time monitoring of overall cell status. To demonstrate this technique, we used three cell lines as sensing probes: two human lung carcinoma cell lines, A549 and SK-MES-1, and a normal mammalian cell line, CHO-K1. We tested two well-characterized nanoparticles: nano-titanium dioxide (nTiO2) and nano-silver (nAg). The three cell lines were separately seeded into 96x E-plates and treated with varying concentrations of nanoparticles (0.078–160μgmL−1). This method provides dynamic cell response profiles and temporal IC50 histograms, showing concentration-, time-, particle-, and cell-dependent cytotoxicity. The 24h and 48h IC50 values of nAg obtained using both the RTCA and the neutral red uptake (NRU) assays were in good agreement, validating the RTCA technique. The RTCA assay does not suffer interference from nTiO2, whereas the NRU assay cannot be used due to severe interference from nTiO2. A cytostatic response was observed in CHO-K1 cells after 24h exposure to 40μgmL−1 nTiO2, which was correlated with S-phase cell cycle arrest based on cell cycle analysis using flow cytometry. This suggests that the shapes of the response curves provide indicative information, directing further studies into the mode of action of the toxicant. Advantages of the RTCA technique over traditional colorimetric assays for screening the cytotoxicity of nanoparticles include minimizing interference, qualitative and quantitative cytotoxicity data, and the capability of real-time and high-throughput measurements.

UPEC biomimickry at the urothelial barrier: Lectin-functionalized PLGA microparticles for improved intravesical chemotherapy

The urgent demand for more potent treatment schedules in bladder cancer (BCa) therapy calls for a refinement of the intravesical administration modalities. However, progress on drug delivery systems tailored to the penetration-hostile urothelial barrier lags behind the advancements in comparable fields. This study reports on a multimodal, carrier-based delivery concept that combines biorecognitive targeting with modified release strategies for improved intravesical chemotherapy. The plant lectin wheat germ agglutinin (WGA) was immobilized on poly(lactide-co-glycolide) (PLGA) microparticles (MP) to induce stable cytoadhesion via cellular carbohydrate chains, similar to the specific attachment mechanism utilized by uropathogenic bacteria. A panel of DNA-selective chemotherapeutics with established track record in uro-oncology was screened for physicochemical compatibility with the polymeric carrier formulation. Critical limitations in encapsulation efficiency were found for mitomycin C (MMC), doxorubicin (DOX), and gemcitabine hydrochloride (GEM), despite multiparametric optimization of the preparation conditions. In contrast, the amphiphilic 4-(N)-stearoyl prodrug of gemcitabine (GEM-C18) exhibited excellent processability with PLGA. In vitro bioassays on 5637 human BCa cells showed that the enhanced cytoadhesion of WGA-GEM-C18-PGLA-MP traces back to the specific lectin/carbohydrate interaction, and is not easily disrupted by adverse environmental factors. Owing to several synergistic effects, the combined prodrug/targeting approach resulted in strong cytostatic response even when adjusting the exposure scheme to the confined temporal conditions of instillative treatment. Our results highlight the importance of fine-tuning both pharmacokinetic and pharmacologic parameters to gain adequate impact on urothelial cancer cells, and assign promising potential to glycan-targeted delivery concepts for the intravesical route.

Endothelial Colony Forming Cells (ECFCs) As a Model for Studying Effects of Low-Dose Ionizing Radiation: Growth Inhibition by a Single Dose

Identification of measurable nontransient responses to low-dose radiation in human primary cell cultures remains a problem. To this end, circulating endothelial colony-forming (progenitor) cells (ECFCs) were examined as an experimental model. ECFCs were isolated from three cord blood donors. Cells were positive for endothelial cell markers and remained highly proliferative after long-term cryopreservation. A single dose of X-ray radiation (0.06–0.38 Gy) inhibited ECFC culture growth. This effect was evident at 48 hours and persisted up to 72 hr postirradiation. Such protracted cytostatic response of ECFCs to low-dose radiation suggests that ECFC primary cultures can be used to study low-dose radiation effects.