mk-2206 and Urinary-Bladder-Neoplasms

KNSTRN is a component of the mitotic spindle, which was rarely investigated in tumorigenesis. AKT plays an essential role in tumorigenesis by modulating the phosphorylation of various substrates. The activation of AKT is regulated by PTEN and PIP

Topics: Apoptosis; Carcinogenesis; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Deoxycytidine; Drug Resistance, Neoplasm; Gemcitabine; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Microtubule-Associated Proteins; Oncogene Protein v-akt; Phosphorylation; Urinary Bladder Neoplasms

The tumor microenvironment is associated with various tumor progressions, including cancer metastasis, immunosuppression, and tumor sustained growth. Tumor-associated macrophages (TAMs) are considered an indispensable component of the tumor microenvironment, participating in the progression of tumor microenvironment remodeling and creating various compounds to regulate tumor activities. This study aims to observe enriched TAMs in tumor tissues during bladder cancer development, which markedly facilitated the proliferation of bladder cancer cells and promoted tumor growth in vivo. We determined that TAMs regulate tumor sustained growth by secreting type I collagen, which can activate the prosurvival integrin α2β1/PI3K/AKT signaling pathway. Furthermore, traditional chemotherapeutic drugs combined with integrin α2β1 inhibitor showed intensive anticancer effects, revealing an innovative approach in clinical bladder cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Chromones; Collagen; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Integrin alpha2beta1; Macrophages; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Microenvironment; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Targeting the PI3K pathway has achieved limited success in cancer therapy. One reason for the disappointing activity of drugs that interfere with molecules that are important player in this pathway is the induction of multiple feedback loops that have been only partially understood. To understand these limitations and develop improved treatment strategies, we comprehensively characterized molecular mechanisms of PI3K pathway signaling in bladder cancer cell lines upon using small molecule inhibitors and RNAi technologies against all key molecules and protein complexes within the pathway and analyzed functional and molecular consequences. When targeting either mTORC1, mTOR, AKT or PI3K, only S6K1 phosphorylation was affected in most cell lines examined. Dephosphorylation of 4E-BP1 required combined inhibition of PI3K and mTORC1, independent from AKT, and resulted in a robust reduction in cell viability. Long-term inhibition of PI3K however resulted in a PDK1-dependent, PIP3 and mTORC2 independent rephosphorylation of AKT. AKT rephosphorylation could also be induced by mTOR or PDK1 inhibition. Combining PI3K/mTOR inhibitors with AKT or PDK1 inhibitors suppressed this rephosphorylation, induced apoptosis, decreased colony formation, cell viability and growth of tumor xenografts. Our findings reveal novel molecular mechanisms that explain the requirement for simultaneous targeting of PI3K, AKT and mTORC1 to achieve effective tumor growth inhibition.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Chick Embryo; Enzyme Inhibitors; Feedback, Physiological; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Mechanistic Target of Rapamycin Complex 1; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivity and possible accelerating cell-killing mechanisms. In this study, we investigated how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in urothelial cancer cells.. We examined bladder cancer cell lines for the expression of phospho(p)-Akt and its downstream targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was examined by transwell invasion assay, and the activities of the Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot.. The expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasive bladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion in UMUC3 cell line and significantly increased the susceptibility of bladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity and apoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressed tumor growth in subcutaneous xenograft models.. These results suggest that concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis in urothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeutic option to enhance chemosensitivity in bladder cancer.

Topics: Allosteric Site; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cisplatin; Drug Synergism; Female; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Signal Transduction; Urinary Bladder Neoplasms; Urothelium

Alterations in the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway are frequent in urothelial bladder cancer (BLCA) and thus provide a potential target for novel therapeutic strategies. We investigated the efficacy of the AKT inhibitor MK-2206 in BLCA and the molecular determinants that predict therapy response.. Biochemical and functional effects of the AKT inhibitor MK-2206 were analysed on a panel of 11 BLCA cell lines possessing different genetic alterations. Cell viability (CellTiter-Blue, cell counts), apoptosis (caspase 3/7 activity) and cell cycle progression (EdU incorporation) were analysed to determine effects on cell growth and proliferation. cDNA or siRNA transfections were used to manipulate the expression of specific proteins such as wild-type or mutant PIK3CA, DUSP1 or CREB. For in vivo analysis, the chicken chorioallantoic membrane model was utilised and tumours were characterised by weight and biochemically for the expression of Ki-67 and AKT phosphorylation.. Treatment with MK-2206 suppressed AKT and S6K1 but not 4E-BP1 phosphorylation in all cell lines. Functionally, only cell lines bearing mutations in the hotspot helical domain of PIK3CA were sensitive to the drug, independent of other genetic alterations in the PI3K or MAPK signalling pathway. Following MK-2206 treatment, the presence of mutant PIK3CA resulted in an increase in DUSP1 expression that induced a decrease in ERK 1/2 phosphorylation. Manipulating the expression of mutant or wild-type PIK3CA or DUSP1 confirmed that this mechanism is responsible for the induction of apoptosis and the inhibition of tumour proliferation in vitro and in vivo, to sensitise cells to AKT target therapy.Conclusion or interpretation:PIK3CA mutations confer sensitivity to AKT target therapy in BLCA by regulating DUSP1 expression and subsequent ERK1/2 dephosphorylation and can potentially serve as a stratifying biomarker for treatment.

Topics: Animals; Apoptosis; Cell Line, Tumor; Chickens; Chorioallantoic Membrane; Class I Phosphatidylinositol 3-Kinases; Dual Specificity Phosphatase 1; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds, 3-Ring; Humans; Molecular Targeted Therapy; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Urinary Bladder Neoplasms