MLK3 phosphorylation by ERK1/2 is required for oxidative stress-induced invasion of colorectal cancer cells

Abstract

Mixed lineage kinase 3 (MLK3) functions in migration and/or invasion of several human cancers; however, the role of MLK3 in colorectal cancer (CRC) invasion is unknown. MLK3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) which activates MAPK pathways through either kinase-dependent or -independent mechanisms. Human colorectal tumors display increased levels of reactive oxygen species (ROS) or oxidative stress. ROS, such as H2O2, are important for carcinogenesis and activate MAPK signaling pathways. In human colorectal carcinoma (HCT116) cells treated with H2O2, extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated and MLK3 exhibited reduced electrophoretic mobility (shift) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was eliminated by phosphatase treatment. Pretreatment with the ROS scavenger N- acetyl-L-cysteine (NAC), the ERK1/2 inhibitor UO126, or ERK1/2 siRNA knockdown blocked the H2O2-induced shift of MLK3, while MLK3 inhibition with Cep1347 did not. In co-immunoprecipitation experiments performed on H2O2-treated HCT116 cells, endogenous MLK3 associated with endogenous ERK1/2 and B-Raf. Active ERK1 phosphorylated kinase dead FLAG-MLK3 in vitro, whereas ERK1 phosphorylation of kinase dead FLAG-MLK3-S705A-S758A was reduced. Both MLK3 siRNA knockdown and FLAG-MLK3-S705A-S758A expression decreased ERK1/2 activation in H2O2-treated cells. Prolonged H2O2 treatment activated ERK1/2 and promoted invasion of colon cancer cells, which was attenuated by MLK3 siRNA knockdown. Furthermore, S705A-S758A-FLAG-MLK3 demonstrated decreased oxidative-stress induced colon cancer cell invasion, but increased interaction with GST-B-Raf as compared to wild-type-FLAG-MLK3 in H2O2-treated cells. These results suggest oxidative stress stimulates an ERK1/2-dependent phosphorylation of MLK3 on Ser705 and Ser758, which promotes MLK3-dependent B-Raf and ERK1/2 activation; this positive feedback loop (PFL) enhances the invasion of colon cancer cells.

MLK3 silence induces cervical cancer cell apoptosis via the Notch‐1/autophagy network

“…MLK3 can activate multiple signal pathways, including the p38 MAPK, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2) MAPK and notch pathway. 8,19 Ample evidence has implicated Notch-1 in autophagy, 20,21 and we speculate that MLK3 may be involved in the regulation of Notch-1 in cervical cancer. In this study, we aim to determine how MLK3 regulates cervical cancer cell apoptosis through the Notch-1/autophagy network.…”

“…MLK3 overexpression occurs in several human tumors, and it is involved in cancer cell proliferation, invasion, and migration. Studies have highlighted its potential role as a relevant therapeutic target for cancer treatments . Schroyer et al and Zhan et al suggested that MLK3 played a critical role in gynaecological malignancies, such as ovarian cancer .…”

“…Gnanadhas et al found that MLK3 inhibitor could induce autophagy in monocyte‐derived macrophages. MLK3 can activate multiple signal pathways, including the p38 MAPK, c‐Jun N‐terminal kinase (JNK), extracellular signal‐regulated kinases 1 and 2 (ERK1/2) MAPK and notch pathway . Ample evidence has implicated Notch‐1 in autophagy, and we speculate that MLK3 may be involved in the regulation of Notch‐1 in cervical cancer.…”

“…The mixed‐lineage kinase (MLK) protein family includes serine, threonine and tyrosine kinase sequences in its catalytic domains . MLKs belong to the mitogen‐activated protein kinase kinase kinase (MAPKKK) family and participate in activation of MAPK signalling pathways in a kinase‐dependent or kinase‐independent manner . Mixed‐lineage kinase 3 (MLK3), also known as MAP3K11, plays a vital role in human diseases, such as cancer .…”

“…6,7 MLKs belong to the mitogen-activated protein kinase kinase kinase (MAPKKK) family and participate in activation of MAPK signalling pathways in a kinase-dependent or kinase-independent manner. 8 Mixed-lineage kinase 3 (MLK3), also known as MAP3K11, plays a vital role in human diseases, such as cancer. 9,10 It also has been implicated in oxidative stress-induced invasion of colorectal cancer cells 8 and the bio-function of cancer cells, such as proliferation, apoptosis, migration, and invasion.…”