dactolisib and Ovarian-Neoplasms

Multi-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX).. Ovarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ.. BEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones.. These results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX.. Dual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.

Topics: ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Ovarian Neoplasms; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH‑2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Ovarian Neoplasms; Protein Kinase Inhibitors; Quinolines; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity.. The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg(-1), 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology.. The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found.. Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235.

Topics: Animals; Antineoplastic Agents; Female; Humans; Imidazoles; Magnetic Resonance Imaging; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Quinolines; Signal Transduction; Treatment Outcome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation (p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis (p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly (p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases.

Topics: Algorithms; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Survival; Chickens; Chorioallantoic Membrane; Cymenes; Drug Screening Assays, Antitumor; Endothelial Cells; Feedback; Female; Humans; Imidazoles; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Organometallic Compounds; Ovarian Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Quinolines; Stochastic Processes

Ovarian mucinous adenocarcinoma (MAC) resists standard chemotherapy and is associated with poor prognosis. A more effective treatment is needed urgently. The present study assessed the possibility of molecular-targeted therapy with a novel dual inhibitor of phosphatidylinositol 3'-kinase (PI3K) and mammalian target of rapamycin (mTOR), NVP-BEZ235 (BEZ235) to treat of MAC. Seven human MAC cell lines were used in this study. The sensitivity of the cells to BEZ235, temsirolimus, and anticancer agents was determined with the WST-8 assay. Cell cycle distribution was assessed by flow cytometry, and the expression of proteins in apoptotic pathways and molecules of the PI3K/Akt/mTOR signaling pathways was determined by Western blot analysis. We also examined the effects of BEZ235 on tumor growth in nude mice xenograft models. The cell lines showed half-maximal inhibitory concentration values of BEZ235 from 13 to 328 nmol/L. Low half-maximal inhibitory concentration values to BEZ235 were observed in MCAS and OMC-1 cells; these 2 lines have an activating mutation in the PIK3CA gene. NVP-BEZ235 down-regulated the protein expression of phosphorylated (p-) Akt, p-p70S6K, and p-4E-BP1, suppressed cell cycle progression, up-regulated the expression of cleaved PARP and cleaved caspase 9, and increased apoptotic cells. Synergistic effects were observed on more than 5 cell lines when BEZ235 was combined with paclitaxel or cisplatin. The treatment of mice bearing OMC-1 or RMUG-S with BEZ235 significantly suppressed tumor growth in MAC xenograft models without severe weight loss. We conclude that the PI3K/Akt/mTOR pathway is a potential therapeutic target and that BEZ235 should be explored as a therapeutic agent for MAC.

Topics: Adenocarcinoma, Mucinous; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Drug Synergism; Female; Humans; Imidazoles; Mice; Mice, Nude; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation; Xenograft Model Antitumor Assays

We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Here we assessed the anticancer potential of combining ABT-737-induced inhibition of Bcl-xL with Mcl-1 inhibition via PI3K/Akt/mTOR pathway disruption using NVP-BEZ235. NVP-BEZ235 inhibited cell proliferation without inducing apoptosis. It strongly repressed Mcl-1 expression and induced Puma expression in both cell lines tested while differentially modulating Bim between the two. Interestingly, NVP-BEZ235 efficiently sensitized ovarian carcinoma cells to ABT-737, provided that Bim expression was induced. Moreover, inhibiting the ERK1/2 pathway restored Bim expression and sensitized low Bim-expressing cancer cells to the BEZ235/ABT-737 treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Ovarian Neoplasms; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Quinolines; RNA Interference; Signal Transduction; Sulfonamides; Time Factors; TOR Serine-Threonine Kinases; Transfection

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Topics: Adenocarcinoma, Clear Cell; Animals; Cell Line, Tumor; Cystadenoma, Serous; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, Nude; Neoplasms, Experimental; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The PI3K/mTOR-pathway is the most commonly dysregulated pathway in epithelial cancers and represents an important target for cancer therapeutics. Here, we show that dual inhibition of PI3K/mTOR in ovarian cancer-spheroids leads to death of inner matrix-deprived cells, whereas matrix-attached cells are resistant. This matrix-associated resistance is mediated by drug-induced upregulation of cellular survival programs that involve both FOXO-regulated transcription and cap-independent translation. Inhibition of any one of several upregulated proteins, including Bcl-2, EGFR, or IGF1R, abrogates resistance to PI3K/mTOR inhibition. These results demonstrate that acute adaptive responses to PI3K/mTOR inhibition in matrix-attached cells resemble well-conserved stress responses to nutrient and growth factor deprivation. Bypass of this resistance mechanism through rational design of drug combinations could significantly enhance PI3K-targeted drug efficacy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Cycle Proteins; Drug Resistance, Neoplasm; ErbB Receptors; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Proto-Oncogene Proteins c-bcl-2; Quinolines; Receptor, IGF Type 1; RNA, Messenger; Signal Transduction; Stress, Physiological; TOR Serine-Threonine Kinases; Transplantation, Heterologous

mTOR is a serine/threonine kinase that acts by binding different sets of proteins forming two complexes, termed mTORC1 and mTORC2. mTOR is deregulated in a substantial proportion of ovarian tumors. Despite the use of drugs directed to mTOR in ongoing clinical trials, the functional relevance of the individual mTORC branches in ovarian cancer is not known. Here, we show that mTORC1 and mTORC2 were constitutively active in ovarian cancer cell lines. Knockdown of raptor or rictor, proteins required for the function of mTORC1 or mTORC2, respectively, resulted in profound inhibition of ovarian cancer cell proliferation. The knockdown of raptor had a more important inhibitory effect than the knockdown of rictor, indicating mTORC1 had a predominant role over mTORC2 in the control of ovarian cancer cell proliferation. Rapamycin decreased the proliferation of ovarian cancer cells, and this was accompanied by inhibition of the phosphorylation of S6, a protein used as readout of mTORC1 function. However, rapamycin had only a marginal effect on the phosphorylation status of 4E-BP1, another mTORC1 substrate. Therefore, mTORC1 probably controls p4E-BP1 along two distinct pathways, one of them sensitive to rapamycin and another insensitive. The dual PI3K/mTOR inhibitor BEZ235 was more efficient than rapamycin in its inhibitory action on ovarian cancer cell proliferation. Biochemically, BEZ235 completely inhibited pS6, p4E-BP1, and pAkt. Our results suggest that broad-spectrum mTOR inhibitors that block mTORC1 and mTORC2 are more desirable for their clinical development in ovarian cancer than agents exclusively targeting one of the mTOR branches.

Topics: Animals; Cell Proliferation; Female; Humans; Imidazoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Ovarian Neoplasms; Protein Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Transplantation, Heterologous

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, and gefitinib), complete growth inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drug-exposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled sensitivity in drug-resistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drug-refractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.

Topics: Aminoquinolines; Aniline Compounds; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; ErbB Receptors; Female; Humans; Imidazoles; Molecular Targeted Therapy; Morpholines; Ovarian Neoplasms; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection

This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy.. Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-ras(G12D/+)Pten(loxP/loxP)).. NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC(50) for NVP-BEZ235. In LSL-K-ras(G12D/+)Pten(loxP/loxP) mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration.. Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intra-abdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer.

Topics: Animals; Antineoplastic Agents; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Disease Models, Animal; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; Imidazoles; Immunoblotting; Mice; Mice, Transgenic; Mutation; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases