dactolisib and Colorectal-Neoplasms

One of the most lethal malignancies worldwide is colorectal cancer (CRC). Alterations in various signalling pathways, including PI3K-mTOR and NF-κB, have been reported in CRC with subsequent dysregulation of proliferation, apoptosis, angiogenesis and, questionably, autophagy processes. BEZ-235 (dactolisib) is a dual PI3K-mTOR inhibitor with potent anti-tumour activity. However, the observed toxicity of BEZ-235 necessitated the termination of its clinical trials. Hence, we aimed to evaluate the potential long-lasting anti-carcinogenic effects of adding diosmin (DIO, a natural NF-κB inhibitor) to BEZ-235 in HCT-116 CRC cells. The median inhibitory concentrations (IC50s) of BEZ-235 and/or DIO were evaluated in the HCT-116 CRC cell line. Caspase-3 activity was assessed colorimetrically, and p-Akt, NF-κB, CD1, VEGF and LC3B levels were assessed by ELISA. Additionally, LC3-II and P62 gene expression were assessed using qRT-PCR. The observed CIs (combination indices) and DRIs (dose reduction indices) confirmed the synergistic effect of DIO and BEZ-235. Co-administration of both drugs either in combination-1 (1 μM for BEZ-235, 250 μM for DIO) or in combination-2 (0.51 μM for BEZ-235 + 101.99 μM for DIO) inhibited the PI3K/Akt/mTOR/NF-κB axis, leading to the induction of apoptosis (via active caspase-3), and the inhibition of proliferation marker (CD1), angiogenesis marker (VEGF), autophagy protein (LC3B) and altered effects on LC3-IIandP62 gene expression. Our results reveal the synergistic chemotherapeutic effects of DIO combined with BEZ-235 in the HCT-116 CRC cell line and encourage future preclinical and clinical studies of this combination with reduced BEZ-235 concentrations to avoid its reported toxicity.

Topics: Cell Proliferation; Cell Survival; Colorectal Neoplasms; Diosmin; Dose-Response Relationship, Drug; Drug Synergism; HCT116 Cells; Humans; Imidazoles; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Therapeutics targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway initially produce potent antitumor effects, but resistance frequently occurs. Using a phosphoproteome analysis, we found that colorectal cancer (CRC) cells exhibit resistance against PI3K/mTOR inhibition through feedback activation of multiple receptor tyrosine kinases, and their downstream focal adhesion kinase, Src and extracellular signal-regulated kinases signaling. Unexpectedly, PI3K/mTOR blockade causes senescence, mediated by the activation of the stress kinase p38. The senescent cancer cells induce the secretion of various cytokines and this senescence-associated secretome increases migration and invasion capabilities of CRC cells. We found that cotargeting PI3K/mTOR and bromodomain and extra-terminal domain can suppress activation of many oncogenic kinases involved in resistance to the PI3K/mTOR inhibition, induce cell death in vitro and tumor regression in vivo, and further prolong the survival of xenograft models. Our findings provide a rationale for a novel therapeutic strategy to overcome resistance to the PI3K/mTOR inhibitors in CRC.

Topics: Animals; Azepines; Caco-2 Cells; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Imidazoles; Mice; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinase; Phosphorylation; Proteomics; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Triazoles; Xenograft Model Antitumor Assays

Drug resistance remains a major clinical problem despite advances in targeted therapies. In recent years, methods to culture cancer cells in three-dimensional (3D) environments to better mimic native tumors have gained increasing popularity. Nevertheless, unlike traditional two-dimensional (2D) cell cultures, analysis of 3D cultures is not straightforward. Most biochemical assays developed for 2D cultures have to be optimized for use with 3D cultures. We addressed this important problem by presenting a simple method of quantitative size-based analysis of growth and drug responses of 3D cultures of cancer cells as tumor spheroids. We used an aqueous two-phase system to form consistently sized tumor spheroids of colorectal cancer cells. Using spheroid images, we computed the size of spheroids over time and demonstrated that growth of spheroids from this analysis strongly correlates with that using a PrestoBlue biochemical assay optimized for 3D cultures. Next, we cyclically treated the tumor spheroids with a MEK inhibitor, trametinib, for 6-day periods with a recovery phase in between. This inhibitor was selected because of mutation of colon cancer cells in the MEK/ERK pathway. We used size measurements to evaluate the efficacy of trametinib and predict development of resistance of colon cancer cells during the cyclical treatment and recovery regimen. This size-based analysis closely matched the biochemical analysis of drug responses of spheroids. We performed molecular analysis and showed that resistance to trametinib emerged due to feedback activation of the PI3K/AKT signaling pathway. Therefore, we combined trametinib with a PI3K/AKT inhibitor, dactolisib, and demonstrated that size-based analysis of spheroids reliably allowed quantifying the effect of the combination treatment to prevent drug resistance. This study established that size measurements of spheroids can be used as a straightforward method for quantitative studies of drug responses of tumor spheroids and identifying drug combinations that block resistance.

Topics: Antineoplastic Agents; Colorectal Neoplasms; Drug Combinations; Drug Resistance, Neoplasm; HCT116 Cells; HT29 Cells; Humans; Imidazoles; Particle Size; Pyridones; Pyrimidinones; Quinolines; Spheroids, Cellular; Tumor Cells, Cultured

The mammalian target of rapamycin (mTOR), a downstream effector of the PI3K/Akt signalling pathway, is a critical regulator of cell metabolism, growth and survival in response to oncogenic factors. Activation of mTOR frequently occurs in human tumours making it a crucial and validated target in the treatment of cancer. mTOR inhibitors such as rapamycin and its analogues decrease cancer progression in experimental models including colorectal cancer (CRC). Recently, the second generation ATP‑competitive mTOR kinase (such as PP242) and dual mTOR/PI3K (such as NVP‑BEZ235) inhibitors have entered clinical trials as anticancer agents. However, in CRC, the efficacy of these novel drugs needs to be fully investigated. In the present study, we examined five human CRC cell lines, HT29, HCT116, SW480, SW620 and CSC480 to evaluate their sensitivity to three mTOR inhibitors, RAD001, PP242 and NVP‑BEZ235. We observed that compared to RAD001 and PP242, NVP‑BEZ235 markedly reduced cell proliferation of CRC cells. Furthermore, we found that the reduced cell proliferation caused by NVP‑BEZ235 was not achieved through the disruption of mitochondrial potential. Using an mTOR‑specific signalling pathway phospho array we revealed that NVP‑BEZ235 significantly decreased phosphorylation of 4E‑BP1 (Thr70), the downstream target of mTORC1. In addition, NVP‑BEZ235 decreased phosphorylation of AKT (Ser473), the downstream target of mTORC2. Immunoblotting analysis revealed that NVP‑BEZ235 effectively inhibited 4E‑BP1 phosphorylation, while PP242 had a weak inhibitory effect. However, PP242 and NVP‑BEZ235 decreased AKT levels in all cell lines. RAD001 demonstrated no effect on 4E‑BP1. Based on the above‑mentioned results, the dual PI3K/mTOR and ATP‑competitive mTOR inhibitors have demonstrated high potential for targeting the mTOR pathway in CRC.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Everolimus; HCT116 Cells; HT29 Cells; Humans; Imidazoles; Indoles; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Autophagy is a conserved lysosomal degradation pathway that regulates cell survival and death in order to maintain cellular homeostasis. Dysfunctional autophagy is associated with different types of cancer, making it an attractive therapeutic target. Mammalian target of rapamycin (mTOR) signaling negatively regulates autophagy and suppresses the efficacy of certain cancer therapeutic agents. NVP-BEZ235 is a dual inhibitor of the PI3K/mTOR signaling pathway and exhibits anti-cancer activities; it also induced autophagy and inhibited proliferation in colorectal adenoma HT-29 cells. Colorectal adenoma and colorectal cancer have been recently shown to have elevated levels of miR-212. In the current study, we examined the role of miR-212 in NVP-BEZ235-induced autophagy in HT-29 cells. NVP-BEZ235 at the concentration as low as of 1 nM effectively induced autophagy and dose-dependently inhibited the expression of microRNA-212 (miR-212) whereas mTOR activator MHY1485 elevated the miR-212 expression. Transfection of miR-212 mimics inhibited autophagy whereas miR-212 inhibitors promoted autophagy as assessed by the LC3B-I conversion to LC3B-II and the expression levels of beclin-1. Furthermore, miR-212 mimics activated mTOR whereas miR-212 inhibitors suppressed mTOR activation as shown by the levels of phospho-mTOR. miR-212 mimics further enhanced the effect of NVP-BEZ235 in reducing the viability of HT-29 cells. Our data support that miR-212 is a target of mTOR signaling as well as an activator of mTOR to negatively regulate autophagy. Thus, miR-212 and mTOR signalings may form a positive regulation loop in maintaining cellular homeostasis. This study warrants further investigation of miR-212 as an effective target of autophagy-based cancer therapeutic strategies.

Topics: Antineoplastic Agents; Autophagy; Cell Survival; Colorectal Neoplasms; HT29 Cells; Humans; Imidazoles; MicroRNAs; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Targeting cellular mitosis in tumor cells is an attractive cancer treatment strategy. Here, we report that B220, a synthetic benzenesulfonamide compound, could represent a new mitotic inhibitor for the treatment of colorectal cancer. We examined the action mechanism of B220 in the colorectal carcinoma HCT116 cell line, and found that treatment of cells with B220 caused cells to accumulate in G2/M phase, with a concomitant induction of the mitotic phase markers, MPM2 and cyclin B1. After 48 h of B220 treatment, cells underwent apoptotic cell death via caspase-3 activation and poly(ADP ribose) polymerase (PARP) cleavage. In addition, B220 inhibits autophagy by blocking conversion of microtubule-associated protein 1 light chain 3 (LC3-I) to LC3-II and inhibiting autophagic flux. Notably, blockade of autophagy by pharmacological inhibition or using an Atg5-targeting shRNA reduced B220-induced cytotoxicity. Conversely, the autophagy inducer NVP-BEZ235 shows a synergistic interaction with B220 in HCT116 cells, indicating autophagy was required for the observed cell death. In summary, these results indicate B220 combined with the induction of autophagy using the dual PI3K/mTOR inhibitor, NVP-BEZ235, might be an attractive strategy for cancer therapy, and provides a framework for further development of B220 as a new therapeutic agent for colon cancer treatment.

Topics: Antimitotic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Colorectal Neoplasms; Cyclin B1; HCT116 Cells; Humans; Imidazoles; Kinesins; Microtubule-Associated Proteins; Mitosis; Quinolines

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chromones; Colorectal Neoplasms; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplastic Stem Cells; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; SOXB1 Transcription Factors; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway plays a significant role in colorectal adenocarcinoma. NVP-BEZ235 (dactolisib) is a novel dual inhibitor of PI3K/mTOR. The effects of NVP-BEZ235 in human colorectal adenocarcinoma are still unclear. In the present study, we aimed to explore the proliferation, migration, apoptosis and autophagy in HT-29 human colorectal adenocarcinoma cells. HT-29 human colorectal adenocarcinoma cells were treated with NVP-BEZ235 (0, 0.001, 0.01, 0.1, 1 and 3 µM) for 24 and 48 h, respectively. Cells were also treated with NVP-BEZ235 (0.1 µM), DDP (100, 300 and 1,000 µM), and NVP-BEZ235 (0.1 µM) combined with DDP (100, 300 and 1,000 µM) respectively, and cultured for 24 h after treatment. MTT assay was utilized to evaluate the effects of NVP-BEZ235 alone or NVP-BEZ235 combined with cis-diamminedichloroplatinum (DDP) on proliferation of HT-29 cells. Cell wound-scratch assay was used detect cell migration. In addition, expression of microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B and LC3B) in HT-29 cells was detected by immunofluorescence at 48 h after NVP-BEZ235 (1 µM) treatment. Expression of proteins involved in cell cycle and proliferation (p-Akt, p-mTOR and cyclin D1), apoptosis (cleaved caspase-3), and autophagy (cleaved LC3B and Beclin-1) were detected by western blot analysis. NVP-BEZ235 inhibited the proliferation and migration of HT-29 human colorectal adenocarcinoma cells. NVP-BEZ235 decreased protein expression of p-Akt, p-mTOR and cyclin D1, and increased protein expression of cleaved caspase-3, cleaved LC3B and Beclin-1 as the concentrations and the incubation time of NVP-BEZ235 increased. In addition, NVP-BEZ235 and DDP had synergic effects in inhibiting cell proliferation and migration. The expression of protein involved in apoptosis (cleaved caspase-3) was higher in drug combination group compared to the NVP-BEZ235 single treatment group. NVP-BEZ235 inhibited the proliferation and migration, and induced apoptosis and autophagy of HT-29 human colorectal adenocarcinoma cells.

Topics: Adenocarcinoma; Apoptosis; Autophagy; Cell Movement; Cell Proliferation; Cisplatin; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Imidazoles; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Although epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab are used widely to treat KRAS wild-type metastatic colorectal cancer (mCRC), patients become resistant by various mechanisms, including KRAS, BRAF, and PIK3CA mutations, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we investigated the mechanisms of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in a KRAS and PIK3CA mutation CRC xenograft model. HCT116 (KRAS (G13D) , PIK3CA (H1047R) mutant) cells were subcutaneously injected into the nude mice. Mice were randomly assigned to treatment with vehicle, cetuximab, AZD6244, BEZ235, or AZD6244 plus BEZ235, for up to 3 weeks; then, all mice were sacrificed, and tumor tissues were subjected to Western blot analysis and immunohistochemical staining. AZD6244 or BEZ235 slightly inhibit tumor growth of HCT116 xenografts, and the combination treatment markedly enhanced their antitumor effects. However, cetuximab had no effect on tumor growth. Western blot analysis and immunohistochemical staining revealed that treatment with AZD6244 or BEZ235 could significantly reduce the phosphorylation level of ERK1/2 or AKT in HCT116 tumor tissues. More interesting, the antiangiogenic effects were substantially enhanced when the agents were combined which may due to the reduced expression of VEGF and matrix metallopeptidase-9 (MMP-9) in tumor tissues. These results suggest that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in CRC harboring with KRAS and PIK3CA mutations. The mechanisms of synergistic antitumor effects may be due to antiangiogenesis.

Topics: Animals; Antibodies, Monoclonal, Humanized; Benzimidazoles; Cetuximab; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Imidazoles; MAP Kinase Kinase 1; Matrix Metalloproteinase 9; Mice; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinolines; ras Proteins; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The present study was aimed to investigate whether combination of molecular targeting therapy, a dual PI3K/mTOR inhibitor (BEZ235), with radiation can enhance the radiosensitivity of colorectal cancer cells (CRC). K-RAS mutant CRC cells (HCT 116 and SW 620) and wild type CRC cells (HT 29) were irradiated with different dose of radiation (0-6 Gy). The synergistic effects of combining radiation with different concentration of BEZ235 (0-10 nM) pretreatment were demonstrated by cell survival assay. When comparing with radiation alone and BEZ235 alone, the combination of BEZ235 pretreatment and radiation resulted in an increased percentage of sub-G1 phase cells, and an increased number of γ-H2AX/cell (DNA double strand breaks). Radiation up-regulated AKT/mTOR signaling pathway, including the activation of phospho (p)-AKT, p-mTOR, p-eIF4E, and p-rpS6; and this activated AKT/mTOR signaling pathway was attenuated by BEZ235 pretreatment. In addition, BEZ235 blocked double strand break repair induced by radiation through attenuating the activation of ATM and DNA-PKcs and sensitized CRC cells to radiation. In vivo model, the tumor size and the expression pattern of p-mTOR, p-eIF4E, and p-rpS6 were significantly decreased in combined group than radiation alone or BEZ235 alone. Our findings indicate that the administration of BEZ235 before radiation enhances the radiotherapeutic effect of CRC cells both in vitro and in vivo.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Blotting, Western; Cell Line, Tumor; Cell Survival; Chemoradiotherapy; Colorectal Neoplasms; DNA Breaks, Double-Stranded; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Female; G1 Phase; HCT116 Cells; Humans; Imidazoles; Immunohistochemistry; Mice, SCID; Microscopy, Confocal; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Radiation Tolerance; Radiation-Sensitizing Agents; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

The objective of tailoring medicines for cancer patients according to the molecular profile of their disease holds great promise for the improvement of cancer therapy. Nevertheless, this approach has been limited, in part, due to the lack of predictive and informative preclinical studies. Herein, we describe an assessment of the therapeutic potential of targeting PI3K/mTOR and MAPK signaling in genetically defined mouse models of colorectal cancer mirroring disease subtypes targeted for novel therapy in the FOCUS4 trial. Our studies demonstrate that dual PI3K/mTOR inhibition is highly effective in invasive adenocarcinoma models characterized by combinatorial mutations in Apc and Pten; Apc and Kras; and Apc, Pten and Kras. MEK inhibition was effective in the combinatorial Apc and Kras setting, but had no impact in either Apc Pten mutants or in Apc Pten Kras triple mutants. Furthermore, we describe the importance of scheduling for combination studies and show that although no additional benefit is gained in Apc Pten mice, combination of PI3K/mTOR and MAPK inhibition leads to an additive benefit in survival in Apc Kras mice and a synergistic increase in survival in Apc Pten Kras mice. This is the first study using robust colorectal cancer genetically engineered mouse models to support the validity of PI3K/mTOR and MEK inhibitors as tailored therapies for colorectal cancer and highlight the potential importance of drug scheduling in the clinic.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Benzimidazoles; Colorectal Neoplasms; Disease Models, Animal; Drug Screening Assays, Antitumor; Genes, APC; Imidazoles; MAP Kinase Kinase Kinases; Mice, Transgenic; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; Quinolines; Tumor Burden

Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).. Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.. Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.. These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Female; Fluorine Radioisotopes; Fluorobenzenes; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; Indoles; Mice, Inbred C57BL; Mice, Nude; Organophosphates; Peptides; Positron-Emission Tomography; Protein Kinase Inhibitors; Quinazolines; Quinolines; Radiopharmaceuticals; Sulfonamides; Tissue Distribution; Xenograft Model Antitumor Assays

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Imidazoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Targeted Therapy; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Quinolines; ras Proteins; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa

Colorectal cancer (CRC) with mutational activation of KRAS is observed frequently. In addition, PIK3CA mutations commonly coexist with KRAS mutations and lead to additive activation of the PI3K/MTOR signaling pathway. Here, we investigated how CRC cells that harbor KRAS and PIK3CA mutations affect sensitivity to inhibition of PI3K/MTOR with NVP-BEZ235 (BEZ235). We selected CRC patient samples and assessed their mutational status. CRC patients with KRAS or PIK3CA mutations show activation of AKT and MTOR, particularly when KRAS and PIK3CA mutations coexist. Suppression of PI3K/MTOR by BEZ235 results in a growth inhibitory effect and enhanced apoptosis via BIM activation in KRAS mutant cells. Mutational activation of KRAS when accompanied by a PIK3CA mutation converges at PI3K/MTOR pathway activation, resulting in resistance to BEZ235. BIM knockdown blocked the apoptotic response to BEZ235 in KRAS mutant cells, suggesting that PI3K inhibition leads to BIM accumulation. Moreover, BEZ235 treatment resulted in induction of FOXO3A activity and its induced transcription of BIM activation, which sensitized cells to cytotoxic agents leading to apoptosis in double mutant cells in vitro and in vivo. Taken together, our data suggest that targeting PI3K/MTOR sensitizes cells to apoptosis, implying that activation of PI3K/MTOR signaling via KRAS or PIK3CA mutation is an important pathway in CRC cell growth. Based on these results, coexistent KRAS and PIK3CA mutations confer resistance to BEZ235 via suppression of BIM-induced apoptosis, suggesting that combined treatment with conventional chemoagents is a potential strategy in the clinic.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Female; Flow Cytometry; Genes, ras; Humans; Imidazoles; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).. PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.. In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC(50) = 9.0-14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).. These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; HCT116 Cells; Humans; Imidazoles; Immunohistochemistry; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction