azd-6244 and Cell-Transformation--Neoplastic

Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.

Topics: Animals; Benzimidazoles; Cell Lineage; Cell Proliferation; Cell Transformation, Neoplastic; Gastric Mucosa; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Humans; Kinetics; Mice, Inbred C57BL; Mice, Transgenic; Proto-Oncogene Proteins p21(ras); Signal Transduction; Stomach; Stomach Neoplasms

Gastric cancer develops in the context of parietal cell loss, spasmolytic polypeptide-expressing metaplasia (SPEM), and intestinal metaplasia (IM). We investigated whether expression of the activated form of Ras in gastric chief cells of mice leads to the development of SPEM, as well as progression of metaplasia.. We studied Mist1-CreERT2Tg/+;LSL-K-Ras(G12D)Tg/+ (Mist1-Kras) mice, which express the active form of Kras in chief cells on tamoxifen exposure. We studied Mist1-CreERT2Tg/+;LSL-KRas (G12D)Tg/+;R26RmTmG/+ (Mist1-Kras-mTmG) mice to examine whether chief cells that express active Kras give rise to SPEM and IM. Some mice received intraperitoneal injections of the Mitogen-activated protein kinase kinase (MEK) inhibitor, selumetinib, for 14 consecutive days. Gastric tissues were collected and analyzed by immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction.. Mist1-Kras mice developed metaplastic glands, which completely replaced normal fundic lineages and progressed to IM within 3-4 months after tamoxifen injection. The metaplastic glands expressed markers of SPEM and IM, and were infiltrated by macrophages. Lineage tracing studies confirmed that the metaplasia developed directly from Kras (G12D)-induced chief cells. Selumetinib induced persistent regression of SPEM and IM, and re-established normal mucosal cells, which were derived from normal gastric progenitor cells.. Expression of activated Ras in chief cells of Mist1-Kras mice led to the full range of metaplastic lineage transitions, including SPEM and IM. Inhibition of Ras signaling by inhibition of MEK might reverse preneoplastic metaplasia in the stomach.

Topics: Animals; Anticarcinogenic Agents; Benzimidazoles; Cell Differentiation; Cell Lineage; Cell Proliferation; Cell Transformation, Neoplastic; Chief Cells, Gastric; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Genetic Predisposition to Disease; Humans; Macrophages; Male; Metaplasia; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Mutation; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Stomach Neoplasms; Time Factors; Transcriptional Activation

HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an impact on viability. However, inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Finally, Ba/F3 cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition. Our results show that HRAS mutations in cancer activate the RAS and mTOR pathways which might serve as a therapeutic option for patients with HRAS mutant tumors.

Topics: Animals; Apoptosis; Benzamides; Benzimidazoles; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Diphenylamine; Humans; Mice, SCID; Mitogen-Activated Protein Kinase Kinases; Mutation; Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01 (7-hydroxystaurosporine), AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor -induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK -/- transformed fibroblasts and suppressed by over expression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor -induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.

Topics: Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Benzimidazoles; Benzodioxoles; Breast Neoplasms; Cell Death; Cell Line, Transformed; Cell Transformation, Neoplastic; Checkpoint Kinase 1; Dasatinib; Female; Fibroblasts; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Protease Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-fyn; Proto-Oncogene Proteins c-yes; Pyrimidines; Quinazolines; Radiation Tolerance; src-Family Kinases; Thiazoles