mk-2206 and Colorectal-Neoplasms

PI3K/AKT/mTOR and RAS/RAF/MEK pathways are frequently dysregulated in colorectal cancer (CRC). We conducted a biomarker-driven trial of the combination of MK-2206, an allosteric AKT 1/2/3 inhibitor, and selumetinib, a MEK 1/2 inhibitor, in patients with CRC to evaluate inhibition of phosphorylated ERK (pERK) and AKT (pAKT) in paired tumor biopsies.. Adult patients with advanced CRC were enrolled in successive cohorts stratified by KRAS mutation status. Initially, 12 patients received oral MK-2206 90 mg weekly with oral selumetinib 75 mg daily in 28-day cycles. Following an interim analysis, the doses of MK-2206 and selumetinib were increased to 135 mg weekly and 100 mg daily, respectively. Paired tumor biopsies were evaluated for target modulation.. Common toxicities were gastrointestinal, hepatic, dermatologic, and hematologic. Of 21 patients enrolled, there were no objective responses. Target modulation did not achieve the pre-specified criteria of dual 70 % inhibition of pERK and pAKT levels in paired tumor biopsies.. Despite strong scientific rationale and preclinical data, clinical activity was not observed. The desired level of target inhibition was not achieved. Overlapping toxicities limited the ability to dose escalate to achieve exposures likely needed for clinical activity, highlighting the challenges in developing optimal combinations of targeted agents.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Biomarkers, Tumor; Colorectal Neoplasms; Female; HCT116 Cells; Heterocyclic Compounds, 3-Ring; Humans; Male; Middle Aged; Tomography; Treatment Outcome; Xenograft Model Antitumor Assays; Young Adult

Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models.. Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area.. In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro.. Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Everolimus; Heterocyclic Compounds, 3-Ring; Humans; Mice, Inbred Strains; Mice, Nude; Proto-Oncogene Proteins c-akt; Spheroids, Cellular; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Circulating tumor cells (CTCs) are cells shed from the primary tumor into the bloodstream. While many studies on solid tumor cells exist, data on CTCs are scarce. The mortality of cancer is mostly associated with metastasis and recent research identified CTCs as initiators of metastasis. The PI3K/AKT/mTOR signaling pathway is an intracellular pathway that regulates essential functions including protein biosynthesis, cell growth, cell cycle control, survival and migration. Importantly, activating oncogenic mutations and amplifications in this pathway are frequently observed in a wide variety of cancer entities, underlining the significance of this signaling pathway. In this study, we analyzed the functional role of the PI3K/AKT/mTOR signaling pathway in the CTC-MCC-41 line, derived from a patient with metastatic colorectal cancer. One striking finding in our study was the strong sensitivity of this CTC line against AKT inhibition using MK2206 and mTOR inhibition using RAD001 within the nanomolar range. This suggests that therapies targeting AKT and mTOR could have been beneficial for the patient from which the CTC line was isolated. Additionally, a dual targeting approach of AKT/mTOR inside the PI3K/AKT/mTOR signaling pathway in the colorectal CTCs showed synergistic effects in vitro. Depending on the phenotypical behavior of CTC-MCC-41 in cell culture (adherent vs. suspension), we identified altered phosphorylation levels inside the PI3K/AKT/mTOR pathway. We observed a downregulation of the PI3K/AKT/mTOR signaling pathway, but not of the RAS/RAF/MAPK pathway, in CTCs growing in suspension in comparison to adherent CTCs. Our results highlight distinct functions of AKT isoforms in CTC-MCC-41 cells with respect to cell proliferation. Knockdown of AKT1 and AKT2 leads to significantly impaired proliferation of CTC-MCC-41 cells in vitro. Therefore, our data demonstrate that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of CTC-MCC-41.

Topics: Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Everolimus; Gene Expression Regulation, Neoplastic; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Neoplastic Cells, Circulating; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

AKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition.. Cellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS.. In vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model.. This MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Enzyme Inhibitors; Heterocyclic Compounds, 3-Ring; Heterografts; Humans; Magnetic Resonance Spectroscopy; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt

Isofraxidin (IF), a natural coumarin compound, has been reported to possess anti-cancer activity in human liver cancer. However, whether IF is involved in the regulation of colorectal cancer tumorigenesis and development has been not well elucidated.. The cell proliferation were assessed by Cell Counting Kit-8 (CCK-8) and colony formation test, respectively. The transwell assays were conducted to estimate cell migration and invasion abilities. Further, cell apoptosis was evaluated by confocal microscopy analysis, flow cytometry detection and TdT-mediated dUTP Nick-End Labeling (TUNEL) method. Western blot were performed to detect the expression of related protein.. Herein, the result indicated that IF remarkably bated cell proliferation in human colorectal cancer cells HT-29 and SW-480 in a dose- and time-dependent manner. In addition, IF treatment showed obvious inhibitory activity to cell colony formation in HT-29 and SW-480 cells. Confocal microscopy analysis and flow cytometry detection revealed that IF dramatically induced cell apoptosis in HT-29 and SW-480 cells compared with the control. And IF markedly decreased the expression of anti-apoptotic protein bcl-2, whereas the expression of pro-apoptotic proteins, including caspase-3, caspase-9 and bax, notably increased in HT-29 and SW-480 cells. Besides, IF blocked Akt pathway via inhibition expression of p-Akt. Furthermore, MK2206, an Akt inhibitor, could inhibit cell colony formation and induced apoptosis. This effect is even more obvious in the presence of MK2206 and IF compared to that of either agent alone.. Together, the present study reports a novel use of IF in mitigating human colorectal cancer proliferation and inducing apoptosis via blockage of Akt pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Coumarins; Drug Synergism; Heterocyclic Compounds, 3-Ring; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Growing evidence indicates that tumor-initiating cells (TICs) are responsible for tumor growth and progression. Conventional chemotherapeutics do not sufficiently eliminate TICs, leading to tumor relapse. We aimed to gain insight into TIC biology by comparing the transcriptome of primary TIC cultures and their normal stem cell counterparts to uncover expression differences.. We established colonosphere cultures derived from the resection of paired specimens of primary tumor and normal mucosa in patients with CRC. These colonospheres, enriched for TICs, were used for differential transcriptome analyses to detect new targets for a TIC-directed therapy. Effects of target inhibition on CRC cells were studied in vitro and in vivo.. Pathway analysis of the regulated genes showed enrichment of genes central to PI3K/AKT and Wnt-signaling. We identified CD133 as a marker for a more aggressive CRC subpopulation enriched with TICs in SW480 CRC cells in an in vivo cancer model. Treatment of CRC cells with the selective AKT inhibitor MK-2206 caused a decrease in cell proliferation, particularly in the TIC fraction, resulting in a significant reduction of the stemness capacity to form colonospheres in vitro and to initiate tumor formation in vivo. Consequently, MK-2206 treatment of mice with established xenograft tumors exhibited a significant deceleration of tumor progression. Primary patient-derived tumorsphere growth was significantly inhibited by MK-2206.. This study reveals that AKT signaling is critical for TIC proliferation and can be efficiently targeted by MK-2206 representing a preclinical therapeutic strategy to repress colorectal TICs.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; Fluorouracil; Gene Expression Profiling; Heterocyclic Compounds, 3-Ring; Humans; Intestinal Mucosa; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Spheroids, Cellular; Transcriptome; Tumor Suppressor Protein p53; Wnt Signaling Pathway

Drug resistance is one of the major hurdles for cancer treatment. However, the underlying mechanisms are still largely unknown and therapeutic options remain limited. In this study, we show that microRNA (miR)-587 confers resistance to 5-fluorouracil (5-FU)-induced apoptosis in vitro and reduces the potency of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicate that miR-587 modulates drug resistance through downregulation of expression of PPP2R1B, a regulatory subunit of the PP2A complex, which negatively regulates AKT activation. Knockdown of PPP2R1B expression increases AKT phosphorylation, which leads to elevated XIAP expression and enhanced 5-FU resistance; whereas rescue of PPP2R1B expression in miR-587-expressing cells decreases AKT phosphorylation/XIAP expression, re-sensitizing colon cancer cells to 5-FU-induced apoptosis. Moreover, a specific and potent AKT inhibitor, MK2206, reverses miR-587-conferred 5-FU resistance. Importantly, studies of colorectal cancer specimens indicate that the expression of miR-587 and PPP2R1B positively and inversely correlates with chemoresistance, respectively, in colorectal cancer. These findings indicate that the miR-587/PPP2R1B/pAKT/XIAP signaling axis has an important role in mediating response to chemotherapy in colorectal cancer. A major implication of our study is that inhibition of miR-587 or restoration of PPP2R1B expression may have significant therapeutic potential to overcome drug resistance in colorectal cancer patients and that the combined use of an AKT inhibitor with 5-FU may increase efficacy in colorectal cancer treatment.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; HCT116 Cells; Heterocyclic Compounds, 3-Ring; Humans; Male; Mice; Mice, Nude; MicroRNAs; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

There is extensive evidence for the role of aberrant cell survival signaling mechanisms in cancer progression and metastasis. Akt is a major component of cell survival-signaling mechanisms in several types of cancer. It has been shown that activated Akt stabilizes XIAP by S87 phosphorylation leading to survivin/XIAP complex formation, caspase inhibition and cytoprotection of cancer cells. We have reported that TGFβ/PKA/PP2A-mediated tumor suppressor signaling regulates Akt phosphorylation in association with the dissociation of survivin/XIAP complexes leading to inhibition of stress-dependent induction of cell survival.. IGF1R-dependent colon cancer cells (GEO and CBS) were used for the study. Effects on cell proliferation and cell death were determined in the presence of MK-2206. Xenograft studies were performed to determine the effect of MK-2206 on tumor volume. The effect on various cell death markers such as XIAP, survivin, AIF, Ezrin, pEzrin was determined by western blot analysis. Graph pad 5.0 was used for statistical analysis. P < 0.05 was considered significant.. We characterized the mechanisms by which a novel Akt kinase inhibitor MK-2206 induced cell death in IGF1R-dependent colorectal cancer (CRC) cells with upregulated PI3K/Akt signaling in response to IGF1R activation. MK-2206 treatment generated a significant reduction in tumor growth in vivo and promoted cell death through two mechanisms. This is the first report demonstrating that Akt inactivation by MK-2206 leads to induction of and mitochondria-to-nuclear localization of the Apoptosis Inducing Factor (AIF), which is involved in caspase-independent cell death. We also observed that exposure to MK-2206 dephosphorylated Ezrin at the T567 site leading to the disruption of Akt-pEzrin-XIAP cell survival signaling. Ezrin phosphorylation at this site has been associated with malignant progression in solid tumors.. The identification of these 2 novel mechanisms leading to induction of cell death indicates MK-2206 might be a potential clinical candidate for therapeutic targeting of the subset of IGF1R-dependent cancers in CRC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Cell Death; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cytoskeletal Proteins; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Proto-Oncogene Proteins c-akt; RNA Interference; Tumor Burden; Xenograft Model Antitumor Assays