azd-6244 and saracatinib

Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Benzodioxoles; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; MAP Kinase Kinase Kinases; Mice; Mice, Nude; Neoplasm Recurrence, Local; Quinazolines; RNA, Small Interfering; src-Family Kinases; Xenograft Model Antitumor Assays

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas, and targeting MEK as a central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose limitations, a reason for the low response to MEK targeting drugs is thought to be the upregulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of the drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352, while efficiently suppressing proliferation, stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly, MEK inhibition results in a significant increase in matrix metalloproteases (MMP)-2 and membrane-type 1-MMP expression. All together, MEK inhibition in melanoma cells induces a 'mesenchymal' phenotype that is characterised by protease-driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are the main regulators of this process, the SRC kinase inhibitor, saracatinib (AZD0530), completely abolished the MEK inhibitor-induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.

Topics: Benzimidazoles; Benzodioxoles; Cell Adhesion; Cell Division; Collagen; Humans; Integrin beta1; MAP Kinase Kinase Kinases; Melanoma; Neoplasm Invasiveness; Protein Kinase Inhibitors; Quinazolines; src-Family Kinases

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