ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

Abstract

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

Memo Is a Copper-Dependent Redox Protein with an Essential Role in Migration and Metastasis

“…Phospholipase C-g, which is stimulated by ErbB2, also converges on Memo and participates in microtubule capture (13). Moreover, we have shown that Memo increases the actin-depolymerizing and actin-severing activity of cofilin (2).…”

“…Memo knockdown decreases directed motility of breast cancer cell lines in Transwell and Dunn chamber assays (1,2,13). We examined the effect of Memo knockdown using wound closure and invasion assays.…”

“…Although the role of Memo in cell migration is well documented (1,2,4,13), nothing is known about its biochemical function. Memo is structurally homologous to a class of bacterial nonheme, iron-dependent dioxygenases (7), but these enzymes [for example, protocatechuate dioxygenase (PD)] hydrolyze catechols, and Memo lacks this activity ( fig.…”

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

“…These genes include HECTD1 (HECT domain containing E3 ubiquitin protein ligase 1), involved in negative regulation of the Wnt-Axin pathway (Tran et al , 2013), MACF1 (micro-tubule-actin crosslinking factor 1) involved in the regulation of cell migration after ErbB2 stimulation (Benseddik et al , 2013), LOX (lysyl oxidase) involved in creating a fibrotic environment favorable to tumor cell colonization (Cox et al , 2013) and SLC1A3 (solute carrier family 1 [glial high affinity glutamate transporter], member 3) involved in the response to wounding. The genes ADCY2 (adenylate cyclase 2 [brain]) involved in signal transduction from membrane receptors, HNMT (histamine N-methyltransferase) involved in histamine metabolism and USP16 (ubiquitin specific peptidase 16) involved in histone deubiquitination and cell cycle were also found to be more expressed in bone marrow mesenchymal cells.…”

iASPP contributes to cortex rigidity, astral microtubule capture and mitotic spindle positioning

“…To analyze microtubules in migrating cells [28][29][30] , SKBr3 cells co-transfected with EGFP-α-tubulin and the indicated siRNA or cDNA were grown on collagen-coated glass coverslips for 48 h and observed 10 min after the addition of 5 nM HRGβ1 (R&D Systems) using the 63X objective (plan Apochromat NA 1.4) of a fluorescence microscope (Zeiss Axiovert 200) driven by MetaMorph 6.3 software. Images were acquired using a CoolSNAP HQ digital camera (Roper) and the percentage of cells showing microtubules extending towards the cell periphery and perpendicular to the cell membrane was calculated in three independent experiments.…”