mk-2206 and Triple-Negative-Breast-Neoplasms

Triple negative breast cancer (TNBC) is an aggressive disease with a 5-y relative survival rate of 11% after distant metastasis. To survive the metastatic cascade, tumor cells remodel their signaling pathways by regulating energy production and upregulating survival pathways. AMP-activated protein kinase (AMPK) and Akt regulate energy homeostasis and survival, however, the individual or synergistic role of AMPK and Akt isoforms during lung colonization by TNBC cells is unknown. The purpose of this study was to establish whether targeting Akt, AMPKα or both Akt and AMPKα isoforms in circulating cancer cells can suppress TNBC lung colonization. Transient silencing of Akt1 or Akt2 dramatically decreased metastatic colonization of lungs by inducing apoptosis or inhibiting invasion, respectively. Importantly, transient pharmacologic inhibition of Akt activity with MK-2206 or AZD5363 inhibitors did not prevent colonization of lung tissue by TNBC cells. Knockdown of AMPKα1, AMPKα2, or AMPKα1/2 also had no effect on metastatic colonization of lungs. Taken together, these findings demonstrate that transient decrease in AMPK isoforms expression alone or in combination with Akt1 in circulating tumor cells does not synergistically reduce TNBC metastatic lung colonization. Our results also provide evidence that Akt1 and Akt2 expression serve as a bottleneck that can challenge colonization of lungs by TNBC cells.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Cell Line, Tumor; Energy Metabolism; Female; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplastic Cells, Circulating; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; RNA Interference; RNA, Small Interfering; Signal Transduction; Triple Negative Breast Neoplasms

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by poor survival. Radiotherapy plays an important role in treating TNBC. The purpose of this study was to determine whether inhibiting the AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) pathways alone or in combination potentiates radiotherapy in TNBC. AMPKα1 and AMPKα2 knockdown diminished cyclin D1 expression and induced G1 cell cycle arrest but did not induce apoptosis alone or in combination with radiotherapy. Next, we analyzed the role of PI3K p85α, p85β, p110α, p110β, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction. Akt1 and p110α knockdown diminished cyclin D1 expression and induced apoptosis. Silencing Akt1 promoted synergistic apoptosis induction during radiotherapy and further reduced survival after radiation. Treatment with the Akt inhibitor, MK-2206 48 h after radiotherapy decreased Akt1 levels and potentiated radiation-induced apoptosis. Together, our results demonstrate that AMPKα, p110α, and Akt1 promote TNBC proliferation and that Akt1 is a key regulator of radiosensitivity in TNBC. Importantly, combining radiotherapy with the pharmacological inhibition of Akt1 expression is a potentially promising approach for the treatment of TNBC.

Topics: AMP-Activated Protein Kinases; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Heterografts; Humans; Phosphatidylinositol 3-Kinase; Radiation Tolerance; Signal Transduction; Triple Negative Breast Neoplasms; X-Rays

Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dasatinib; Drug Resistance, Neoplasm; Female; Heterocyclic Compounds, 3-Ring; Humans; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC) cells frequently exhibit activated growth factor signaling and resistance to inhibitors for epidermal growth factor receptor (EGFR), despite the overexpression of EGFR protein, and this is associated with a malignant behavior and a poor prognosis. In this study, to elucidate the underlying mechanisms of resistance to EGFR inhibitor and identify inhibitors that exert a synergistic effect with EGFR inhibition, we examined the inhibitory effects of selected protein kinase inhibitors (PKIs) in combination with gefitinib on the viability of a mesenchymal stem-like (MSL) subtype TNBC cell line. MK‑2206, an AKT inhibitor, and a group of mammalian target of rapamycin (mTOR) inhibitors were found to exert synergistic lethal effects in combination with gefitinib in MDA‑MB‑231 cells. The combination of gefitinib/MK‑2206 exerted a prominent synergistic lethal effect in an MTT cell viability assay and a growth inhibitory effect in a long-term colony-forming assay in 2 MSL subtype TNBC cell lines (MDA‑MB‑231 and HS578T) and one basal-like (BL) subtype TNBC cell line (MDA‑MB-468). Gefitinib/MK‑2206 treatment synergistically decreased the mTOR signaling target substrates along with the downregulation of ribosomal protein S6 (RPS6), a marker of cell proliferation and target substrate of the AKT-mTOR signaling pathway. In addition, gefitinib markedly reduced the viability of MDA‑MD‑231 and HS578T cells when regulatory-associated protein of mTOR (RPTOR) was suppressed by siRNA-based knockdown (KD). These results thus suggest that RPTOR mediates, at least partially, the resistance to EGFR inhibition in TNBC cells. Therefore, targeting the mTOR complex 1 (mTORC1) pathway may be a potential strategy for the treatment of EGFR-resistant TNBC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; ErbB Receptors; Female; Gefitinib; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms